Enzyme-linked immunosorbent assay for the pyrethroid deltamethrin

Article abstract of DOI:10.1021/jf0207629

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of deltamethrin was developed. Two haptens, cyano[3-(4-aminophenoxy)phenyl]methyl 1 R-cis-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropanecarboxylate and 3-[(±)-cyano[1R-cis-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropan ecarbonyloxy]methyl] phenoxyacetic acid, were synthesized and conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The assay that was the most sensitive for deltamethrin was optimized and characterized. The /₅₀ for deltamethrin was 17.5 ± 3.6 μg/L, and the lower detection limit was 1.1 ± 0.5 μg/L. This ELISA assay had relatively low cross-reactivities with other major pyrethroids, such as permethrin, phenothrin, bioresmethrin, cyfluthrin, and cypermethrin. Methanol was found to be the best organic cosolvent for this ELISA, with optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters were unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths strongly suppressed the absorbances. To increase the sensitivity of the overall method, a C₁₈ sorbent-based solid-phase extraction was used for river water samples. River water samples fortified with deltamethrin were analyzed according to this method. Good recoveries and correlation with spike levels were observed.

Full text of DOI:10.1021/jf0207629

5526 J. Agric. Food Chem. 2002, 50, 5526−5532
Enzyme-Linked Immunosorbent Assay for the Pyrethroid
Deltamethrin
HU-JANG LEE, GUOMIN SHAN, TAKAHO WATANABE, DONALD W. STOUTAMIRE,
SHIRLEY J. GEE, AND BRUCE D. HAMMOCK*
Department of Entomology and Cancer Research Center, University of California,
Davis, California 95616
A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of deltamethrin was
developed. Two haptens, cyano[3-(4-aminophenoxy)phenyl]methyl 1R-cis-3-(2,2-dibromoethenyl)-
2,2-dimethylcyclopropanecarboxylate and 3-[(()-cyano[1R-cis-3-(2,2-dibromoethenyl)-2,2-dimeth-
ylcyclopropan ecarbonyloxy]methyl]phenoxyacetic acid, were synthesized and conjugated with
thyroglobulin as immunogens. Four antisera were generated and screened against six different coating
antigens. The assay that was the most sensitive for deltamethrin was optimized and characterized.
The I50 for deltamethrin was 17.5 ( 3.6 µg/L, and the lower detection limit was 1.1 ( 0.5 µg/L. This
ELISA assay had relatively low cross-reactivities with other major pyrethroids, such as permethrin,
phenothrin, bioresmethrin, cyfluthrin, and cypermethrin. Methanol was found to be the best organic
cosolvent for this ELISA, with optimal sensitivity observed at a concentration of 40% (v/v). The assay
parameters were unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths strongly
suppressed the absorbances. To increase the sensitivity of the overall method, a C18 sorbent-based
solid-phase extraction was used for river water samples. River water samples fortified with deltamethrin
were analyzed according to this method. Good recoveries and correlation with spike levels were
observed.
KEYWORDS: Deltamethrin; ELISA; environmental monitoring; pyrethroid
INTRODUCTION
Because pyrethroids are a group of highly potent insecticides
with relatively low mammalian toxicity, they are being widely
used in agriculture, forestry, horticulture, public health, and
households throughout the world (1). These compounds have
improved physical and chemical properties and biological
activity compared to their natural analogues. Pyrethroids are
the most potent lipophilic insecticides (2). They have been
detected as surface water contaminants, and impacts to the
environment leading to effects on ecosystem health have been
reported. Many of the toxicological studies on pyrethroids
focused on nontarget invertebrates and aquatic animals (3-5).
Deltamethrin (Figure 1) is a photostable pyrethroid with an
insecticidal activity 10 times greater than that of permethrin on
several major pests (6). Its greater stability to degradation than
that of alternative pyrethroids has made it attractive for uses
requiring longer residual activity (7). Thus, a sensitive, selective,
and rapid method for monitoring residue levels of deltamethrin
is needed, particularly in aquatic ecosystems. Most synthetic
pyrethroids are marketed as mixtures of optical and geometrical
isomers. In contrast, deltamethrin is 1R-cis- in the dibromo-
chrysanthemate moiety and S at the R-cyano carbon.
Figure 1. Structure of deltamethrin.
Current analytical methods for deltamethrin involve multistep
sample cleanup procedures followed by gas chromatography
(GC) and detection by electron capture (GC-EC) or high-
performance liquid chromatography-mass spectrometry (HPLC-
MS) (2, 6, 8, 9). These methods are relatively intensive, time-
consuming, and expensive, and not particularly suitable for large
numbers of samples. An immunoassay would provide a sensi-
tive, selective, and rapid method for the detection of this
pyrethroid at trace levels (10-16).
To raise antibodies that would be selective for deltamethrin,
the deltamethrin molecule must be modified to attach a spacer
arm containing a functional group to facilitate coupling to a
carrier protein. A monoclonal antibody-based assay was devel-
oped using a hapten in which a spacer that terminated in a
carboxylic acid was added to the phenoxybenzyl moiety. This
assay reported an I50 of ∼500 µg/L with a high degree of
selectivity because there was no cross-reactivity with any
pyrethroids tested (17). Another approach was to replace the
R-cyano group with a spacer that terminated in a carboxylic
* Corresponding author [telephone (530) 752-7519; fax (530) 752-1537;
e-mail bdhammock@ucdavis.edu].
10.1021/jf0207629 CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/22/2002

Deltamethrin Immunoassay
J. Agric. Food Chem., Vol. 50, No. 20, 2002 5527
Figure 2. Scheme for hapten synthesis.
Hapten Synthesis and Verification. Syntheses of the haptens were
carried out as outlined in Figure 2. All reactions were straightforward
and followed procedures employed in preceding publications (12-14,
23). NMR spectral data supported all structures, and mass spectra further
confirmed the structures of the final haptens.
acid. Antibodies to this hapten resulted in an assay with an I50
of 130 µg/L, although treatment of sample with alkali to
isomerize the deltamethrin increased the sensitivity of the assay
to 4 µg/L (18, 19). The antibodies reported here were developed
from a novel deltamethrin hapten and resulted in an assay that
is ∼10 times more sensitive than previously reported for
unisomerized deltamethrin.
Cyano[3-(4-nitrophenoxy)phenyl]methyl 1R-cis-3-(2,2-dibromoet-
henyl)-2,2-dimethylcyclopropanecarboxylate (2). A solution of 1R-cis-
3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropanecarboxylic acid (300
mg, 1 mmol) in chloroform (0.3 mL) was treated with thionyl chloride
(145 µL, ∼2 mmol) and dimethyl formamide (0.2 µL) and heated in
an oil bath at 60-65 °C for 1 h. Solvent and excess thionyl chloride
were briefly stripped, then 0.5 mL of hexane was added, and the mixture
was restripped to leave the acid chloride as a yellow oil. Meanwhile,
zinc iodide (6 mg) was added to a solution of 3-(4-nitrophenoxy)-
benzaldehyde (24) (245 mg, 1 mmol) in chloroform (0.6 mL) under
nitrogen in a septum-capped vial. The mixture was chilled in ice, the
bath was removed, and half of a sample of cyanotrimethylsilane (148
µL, 1.1 mmol) was injected. After a mild exotherm, the mixture was
again cooled, and the remaining cyanotrimethylsilane was injected. After
2.5 h at ambient temperature, the reaction mixture was added to a
mixture of glyme (3 mL) and 3 N HCl (0.75 mL). The mixture was
stirred vigorously for 5 min, diluted with water (3 mL), and extracted
twice with butyl chloride. The organic phase was dried (MgSO4) and
stripped to give the cyanohydrin as an oil.
MATERIALS AND METHODS
Chemicals and Immunoreagents. 1R-cis-3-(2,2-Dibromoethenyl)-
2,2-dimethylcyclopropanecarboxylic acid was kindly supplied by The
Institute of Arable Crops Research (Rothamsted Experimental Station,
Long Ashton, U.K.). A number of synthetic routes to this acid are
described in the literature (20-22). Haptens 12 and 13 were made in
this laboratory previously (14). Other organic starting materials for
hapten synthesis were purchased from Aldrich Chemical Co. (Milwau-
kee, WI) and Fisher Scientific (Pittsburgh, PA). Thin-layer chroma-
tography (TLC) was performed using 0.2 mm precoated silica gel 60
F254 on glass plates from E. Merck (Darmstadt, Germany), and
detection was made by ultraviolet (UV) light or iodine vapor stain.
Flash chromatographic separations were carried out on Baker silica
gel (40µm average particle size) using the indicated solvents, where
the f notation denotes a stepwise concentration gradient.
A solution of the cyanohydrin in chloroform (1 mL) was stirred in
an ice bath, and the acid chloride in chloroform (0.5 mL) was added,
followed by a solution of pyridine (100 µL, 1.25 mmol) in chloroform
(0.3 mL) injected over 5 min. After 30 min at ambient temperature,
the mixture was washed with water, 1 N HCl, and sodium bicarbonate
solution, dried (MgSO4), and stripped to the heavy oil. This was flash
chromatographed on silica gel (13 g) (10 f 100% methylene chloride
in hexane), and fractions containing product were combined and stripped
to yield 535 mg (95%) of 2 as a viscous almost colorless oil. Multiple
development of a TLC with 5% glyme in hexane separated the two
diastereoisomers. A 52 mg sample of 2 was separated by radial
chromatography. The sample was injected in 50% butyl chloride in
hexane and then eluted with 5% glyme in hexane to recover 26 mg of
the higher Rf isomer [1H NMR (CDCl3) δ 1.29 (s, 3 H, CH3), 1.32 (s,
3 H, CH3), 1.92 [d, J ) 8.4 Hz, 1 H, CHC(O)], 2.06 (dd, J ) 8.4, 8.4
Hz, 1 H, CdCCH), 6.39 (s, 1 H, CHCN), 6.67 (d, J ) 8.4 Hz, 1 H,
CdCH), 7.04-8.26 (m, 8 H, Ar)] and 26 mg of the lower Rf isomer
[1H NMR (CDCl3) δ 1.21 (s, 3 H, CH3), 1.26 (s, 3 H, CH3), 1.92 [d,
J ) 8.4 Hz, 1 H, CHC(O)], 2.10 (dd, J ) 8.3, 8.3 Hz, 1 H, CdCCH),
6.42 (s, 1 H, CHCN), 6.68 (d, J ) 8.2 Hz, 1 H, CdCH), 7.03-8.26
(m, 8H, Ar).
The coupling reagents were purchased from Aldrich. Goat anti-rabbit
(GAR) immunoglobulin conjugated to horseradish peroxidase (HRP),
bovine serum albumin (BSA), thyroglobulin, Tween 20, and 3,3′,5,5′-
tetramethylbenzidine (TMB) were purchased from Sigma Chemical Co.
(St. Louis, MO).
Instruments. NMR spectra were obtained using a General Electric
QE-300 spectrometer (Bruker NMR, Billerica, MA). Chemical shift
values are given in parts per million (ppm) downfield from internal
tetramethylsilane. Radial chromatographic separations were carried out
on a Chromatotron apparatus (Harrison Research, Inc., Palo Alto, CA),
using 2 mm silica gel plates. Fast atom bombardment high-resolution
mass spectra (FAB-HRMS) were obtained on a ZAB-2SE mass
spectrometer (VG Analytical, Wythenshawe, U.K.), using high-energy
cesium ions at a density flux of 1-2 mA and 35-38 kV to generate
secondary [MH]+ ions. The liquid matrix was a 1:1 mixture of glycerol
or 3-nitrobenzyl alcohol, and cesium iodide was used for mass
calibration at a dynamic resolution of 5000:1. ELISA experiments were
performed in 96-well microplates (Nunc, Roskilde, Denmark), and the
absorbances were measured with a Vmax microplate reader (Molecular
Devices, Menlo Park, CA) in dual-wavelength mode (450-650 nm).

5528 J. Agric. Food Chem., Vol. 50, No. 20, 2002
Lee et al.
Cyano[3-(4-aminophenoxy)phenyl]methyl 1R-cis-3-(2,2-dibromoet-
henyl)-2,2-dimethylcyclopropanecarboxylate (3). Stannous chloride (345
mg, 1.5 mmol) was added to a solution of 2 (168 mg, 0.305 mmol) in
ethanol (1.5 mL). The mixture was stirred under nitrogen and heated
at 70-75 °C for 35 min. The mixture was cooled and diluted with
water, and ether and Celite (0.4 g) were added, followed by solid
NaHCO3 (260 mg) in portions. The resulting mixture was filtered, and
solids were washed with ethyl acetate. The combined organics were
water washed and stripped to a dark oil. A TLC indicated complete
reaction to one major product spot that rapidly darkened on exposure
to light. This oil was flash chromatographed on silica gel (6 g) (25 f
100% CH2Cl2 in hexane followed by 2 f 10% EtOAc in CH2Cl2).
Stripping fractions containing pure product gave 101 mg (64%) of 3
as a pale yellow oil: 1H NMR (CDCl3) δ 1.21 (s, 3 H, CH3), 1.25 (s,
3 H, CH3), 1.30 (s, 3 H, CH3), 1.31 (s, 3 H, CH3), 1.90 [d, J ) 8.4 Hz,
1 H, CHC(O)], 1.91 [d, J ) 8.4 Hz, 1 H, CHC(O)], 2.03 (t, J ) 8.4
Hz, 1 H, CdCCH), 2.07 (t, J ) 8.4 Hz, 1 H, CdCCH), 3.66 (bs, 2 ×
2 H, NH2), 6.28 (s, 1 H, CHCN), 6.34 (s, 1 H, CHCN), 6.67-7.38 (m,
2 × 8 H, Ar). The CdCH doublets are shifted downfield into the 6.65-
6.67 ppm region and are overlapped by the NH2 doublets. The italicized
values are tentatively assigned to the diastereoisomer corresponding
to the benzyl ester precursor having the higher Rf by TLC. FAB-HRMS
m/z calcd for [M]+ ) C22H20Br2N2O3 was 517.9841 (obsd 518.9844).
Benzyl 3-[(()-Cyano[1R-cis-3-(2,2-dibromoethenyl)-2,2-dimethyl-
cyclopropanecarbony loxy]methyl]phenoxyacetate (5). A sample of
benzyl 2-(3-formylphenoxy)acetate (4) (20) (273 mg, 1.0 mmol) was
converted to the cyanohydrin as a colorless oil as described for 2 above.
This was reacted as described above with 3-(2,2-dibromoethenyl)-2,2-
dimethylcyclopropanecarbonyl chloride prepared from the acid (300
mg, 1.0 mmol) as described above. Flash chromatography of the
resulting product recovered 525 mg (90%) of 5 as a viscous oil. A 51
mg sample was separated by radial chromatography as described above
to recover 25 mg of the higher Rf isomer [1H NMR (CDCl3) δ 1.29 (s,
3 H, CH3), 1.31 (s, 3 H, CH3), 1.90 [d, J ) 8.4 Hz, 1 H, CHC(O)],
2.04 (dd, J ) 8.4, 8.4 Hz, 1 H, CdCCH), 4.70 [s, 2 H, CH2C(O)],
5.25 (s, 2H, CH2Ph), 6.31 (s, 1 H, CHCN), 6.70 (d, J ) 8.4 Hz, 1 H,
CdCH), 6.95-7.40 (m, 9 H, Ar)] and 25 mg of the lower Rf isomer
[1H NMR (CDCl3) δ 1.21 (s, 3 H, CH3), 1.24 (s, 3 H, CH3), 1.90 [d,
J ) 8.4 Hz, 1 H, CHC(O)], 2.07 (dd, J ) 8.4, 8.4 Hz, 1 H, CdCCH),
4.70 [s, 2 H, CH2C(O)], 5.25 (s, 2 H, CH2Ph), 6.36 (s, 1 H, CHCN),
6.71 (d, J ) 8.3 Hz, 1 H, CdCH), 6.94-7.41 (m, 9 H, Ar)].
3-[(()-Cyano[1R-cis-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopro-
panecarbon yloxy]methyl]phenoxyacetic Acid (6). Iodotrimethylsilane
(43 µL, 0.3 mmol) was injected into a solution of 5 (144 mg, 0.25
mmol) in methylene chloride (0.3 mL) contained in a septum-capped
nitrogen-filled test tube. The mixture was warmed in an oil bath at 35
°C for 2 h and then diluted with methanol (1 mL) and methylene
chloride (2 mL). The resulting mixture was water washed and stripped
to a brown oil. This was immediately flash chromatographed on silica
gel (5 g) eluting with 50%CH2Cl2 in hexane (10 mL), 2% EtOAc in
CH2Cl2 (10 mL), and then 20 f 100% EtOAc in CH2Cl2 containing
1.5% v/v HOAc. Fractions containing pure product were stripped, and
EtOAc (5 mL) was added and restripped to remove traces of acetic
acid to yield 94 mg (75%) of 6 as a colorless gum: 1H NMR (CDCl3)
δ 1.22 (s, 3 H, CH3), 1.26 (s, 3 H, CH3), 1.30 (s, 3 H, CH3), 1.32 (s,
3 H, CH3), 1.92 (×2) [d, J ) 8.4 Hz, 2 × 1 H, CHC(O)], 2.05 (dd, J
) 8.4, 8.4 Hz, 1 H, CdCCH), 2.09 (dd, J ) 8.4, 8.4 Hz, 1 H, Cd
CCH), 4.74 (×2) [s, 2 × 2 H, CH2C(O)], 6.35 (s, 1 H, CHCN), 6.39
(s, 1 H, CHCN), 6.70 (d, J ) 8.4 Hz, CdCH), 6.71 (d, J ) 8.4 Hz,
CdCH), 6.98-7.44 (m, 2 × 4 H, Ar). The italicized values are
tentatively assigned to the diastereoisomer corresponding to the benzyl
ester precursor having the higher Rf by TLC. FAB-HRMS m/z calcd
for [M + H]+ ) C18H18Br2NO5 was 485.9552 (obsd 485.9547).
(()-Cyano[3-(4-nitrophenoxy)phenyl]methyl (()-cis-3-(2,2-Dichlo-
roethenyl)-2,2-dimethylcyclopropanecarboxylate (8). Using the same
procedures as those described above for the preparation of 3, (()-cis-
3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarbonyl chloride was
prepared from the corresponding acid [(()-cis-permethric acid] (0.75
g, 3.59 mmol), and the cyanohydrin was prepared from 3-(4-nitrophe-
noxy)benzaldehyde (0.875 g, 3.59 mmol). These were coupled as
described above to yield the crude ester as an oil. Flash chromatography
on silica gel (25 g) (5 f 80% CH2Cl2 in hexane) gave 1.06 g (64%)
of the pure isomer mix, 8, as a colorless gum. A 54 mg sample of 8
was separated by radial chromatography into the two diastereomer pairs
as described for 2 above to return 33.9 mg of the higher Rf isomer pair
[1H NMR (CDCl3) δ 1.29 (s, 3 H, CH3), 1.31 (s, 3 H, CH3), 1.91 (d,
J ) 8.4 Hz, 1 H, CHCOO), 2.14 (dd, J ) 8.6, 8.6 Hz, 1 H, CdCCH),
6.15 (d, J ) 8.8 Hz, 1 H, CdCH), 6.39 (s, 1 H, CHCN), 7.04-8.26
(m, 8 H, Ar); 13C NMR (CDCl3) δ 14.7, 28.1, 29.0, 31.0, 33.5, 62.0,
115.8, 117.5 (2 C), 119.6, 121.9, 122.0, 123.7, 124.3, 126.0 (2 C),
131.2, 134.2, 143.1, 155.5, 162.3, 168.4] and 19.8 mg of the lower Rf
isomer pair [1H NMR (CDCl3) δ 1.21 (s, 3 H, CH3), 1.26 (s, 3 H,
CH3), 1.92 (d, J ) 8.4 Hz, 1 H, CHCOO), 2.18 (dd, J ) 8.6, 8.6 Hz,
1 H, CdCCH), 6.16 (d, J ) 8.5 Hz, 1 H, CdCH, 6.41 (s, 1 H, CHCN),
7.04-8.27 (m, 8 H, Ar); 13C NMR (CDCl3) δ 14.8, 28.1, 28.9, 30.9,
33.6, 62.0, 115.7, 117.6 (2 C), 119.6, 122.0, 122,2, 123.5, 124.4, 126.1
(2 C), 131.2, 134.4, 143.2, 155.5, 162.4, 168.4].
(()-Cyano[3-(4-nitrophenoxy)phenyl]methyl (()-trans-3-(2,2-Dichlo-
roethenyl)-2,2-dimethylcyclopropanecarboxylate (9). This material was
prepared on the same scale and by the same procedure as described
above for 8 using (()-trans-permethric acid to yield 1.06 g (64%) of
the pure isomer mix, 9, as a colorless gum. A 70 mg sample of 9 was
separated by radial chromatography as described above for 2 to yield
45 mg of the higher Rf isomer pair [1H NMR (CDCl3) δ 1.24 (s, 3 H,
CH3), 1.34 (s, 1 H, CH3), 1.67 (d, J ) 5.3 Hz, 1 H, CHCOO), 2.29
(dd, J ) 5.3, 8.1 Hz, 1 H, CdCCH), 5.61 (d, J ) 8.1 Hz, 1 H, Cd
CH), 6.44 (s, 1 H, CHCN), 7.03-8.27 (m, 8 H, Ar); 13C NMR (CDCl3)
δ 19.9, 22.3, 30.5, 33.7, 34.0, 62.3, 115.8, 117.5 (2 C), 119.6, 122.0,
123.1, 124.4, 125.9, 126.0 (2 C), 131.2, 134.2, 143.1, 155.5, 162.3,
169.2] and 25 mg of the lower Rf isomer pair [1H NMR (CDCl3) δ
1.20 (s, 3 H, CH3), 1.25 (s, 3 H, CH3), 1.69 (d, J ) 5.3 Hz, 1 H,
CHCOO), 2.33 (dd, J ) 5.3, 8.1 Hz, 1 H, CdCCH), 5.63 (d, J ) 8.1
Hz, 1 H, CdCH), 6.44 (s, 1 H, CHCN), 7.02-8.25 (m, 8 H, Ar); 13C
NMR (CDCl3) δ 20.0, 22.4, 30.3, 33.7, 34.0, 62.3, 115.6, 117.6 (2 C),
119.5, 122.0, 123.3, 124.3, 125.8, 126.0 (2 C), 131.2, 134.4, 143.2,
155.5, 162.3, 169.2].
(()-Cyano[3-(4-aminophenoxy)phenyl]methyl (()-cis-3-(2,2-Dichlo-
roethenyl)-2,2-dimethylcyclopropanecarboxylate (10). A 5 mL reaction
flask with a magnetic stir bar was charged with compound 8 (250 mg,
0.54 mmol), ethanol (2 mL), and stannous chloride dihydrate (612 mg,
2.7 mmol). The mixture was stirred and heated in an oil bath at 70 °C
for 30 min. The mixture was cooled and poured into a slurry of water,
ether, and Celite (0.45 g). NaHCO3 (0.455 g, 5.4 mmol) was added in
portions (foaming). The neutral mixture was filtered, and solids were
washed with water and ether. The ether phase was separated, dried
(Na2SO4), and stripped to a dark oil. Flash chromatography on 5.5 g
of silica gel (50 f 100% CH2Cl2 in hexane) recovered 16 mg (6.4%)
of 8 and 129 mg (59% based on recovered starting material) of isomer
mix 10 as a stripped gum, Rf 0.15 (silica gel, CH2Cl2). FAB-HRMS
m/z calcd for [M]+ ) C22H20Cl2N2O3 was 430.0851 (obsd 430.0854).
1H NMR (CDCl3) confirmed a mixture of two isomer pairs in a ∼3:2
ratio: δ 1.20 (s, 3 H, CH3), 1.25 (s, 3 H, CH3), 1.29 (s, 3 H, CH3),
1.30 (s, 3 H, CH3), 1.89 (d, J ) 8.4 Hz, 2 × 1 H, CHCO2), 2.09-2.15
(m, 2 × 1 H, CdCCH), 3.6 (bs, 2 × 2 H, NH2), 6.18 (d, J ) 8.8 Hz,
1 H, CdCH), 6.19 (d, J ) 8.8 Hz, 1 H, CdCH), 6.29 (s, 1 H, CHCN),
6.34 (s, 1 H, CHCN), 6.69-7.38 (m, 2 × 8 H, Ar). The italicized peak
values are the more intense of the pair.
(()-Cyano[3-(4-aminophenoxy)phenyl]methyl (()-trans-3-(2,2-
Dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate (11). This mate-
rial was prepared from 9 on the same scale and by the same procedures
as for the preparation of 10 above to yield 128 mg (55%) of the isomer
mix 11 as a gum, Rf 0.15 (silica gel, CH2Cl2). FAB-HRMS m/z calcd
1
for [M]+ ) C22H20Cl2N2O3 was 430.0851 (obsd 430.0852). H NMR
(CDCl3) confirmed a mixture of two isomer pairs in a ∼3:2 ratio: δ
1.18 (s, 3 H, CH3), 1.22 (s, 3 H, CH3), 1.24 (s, 3 H, CH3), 1.32 (s, 3
H, CH3), 1.65 (d, J ) 5.4 Hz, 1 H, CHCO2), 1.67 (d, J ) 5.3 Hz, 1 H,
CHCO2), 2.25-2.33 (m, 2 × 1 H, CdCCH), 3.64 (bs, 2 × 2 H, NH2),
5.60 (d, J ) 7.9 Hz, 1 H, CdCH), 5.62 (d, J ) 7.9 HZ, 1 H, CdCH),
6.34 (s, 1 H, CHCN), 6.36 (s, 1 H, CHCN), 6.68-7.37 (m, 2 × 8 H,
Ar). The italicized peak values are the more intense of the pair.
Hapten Conjugation. Conjugates were synthesized using a water-
soluble carbodiimide or diazotization methods (25, 26). To obtain

Deltamethrin Immunoassay
J. Agric. Food Chem., Vol. 50, No. 20, 2002 5529
immunogens, haptens 3 and 6 were conjugated to thyroglobulin. Coating
antigens were made by coupling haptens 3, 6, 10, 11, 12, and 13 to
BSA.
the synthesis of permethrin haptens in a previous publication
(14). Deltamethrin is a potent pyrethroid insecticide containing
three chiral centers. The commercial material is produced
through a chiral synthesis scheme by which the acid moiety
has the more active 1R-cis configuration and the cyanohydrin
center the S configuration. Because racemization of the cyano-
hydrin center is known to occur under some field conditions
(27), the haptens were synthesized as mixtures of the two
diastereoisomers racemic at the cyanohydrin center to allow
detection of both of these isomers. Although separation of the
immediate precursors was possible as demonstrated under
Materials and Methods, no attempt was made to prepare the
pure chiral isomers or diastereoisomers of the final haptens in
this study.
Hapten 3, 10, or 11 (0.05 mmol) was dissolved in 4 drops of ethanol
and treated with 0.6 mL of 1 N HCl. The resulting solution was stirred
in an ice bath as 0.4 mL of 0.20 M sodium nitrite was added. DMF
(0.4 mL) was then added dropwise to give a homogeneous solution.
Forty-five milligrams of thyroglobulin or BSA was dissolved in a
mixture of 5 mL of 0.2 M borate buffer (pH 8.8) and 1.2 mL of DMF.
The activated hapten solution was added dropwise to the two stirred
protein solutions. The reaction mixtures were stirred in an ice bath for
45 min and then dialyzed against phosphate-buffered saline (PBS) over
72 h at 4 °C. The purified conjugates were suspended in water and
stored in aliquots at -20 °C.
Haptens 6, 12, and 13 were conjugated according to ref 14.
Immunization and Antiserum Preparation. Deltamethrin antisera
were obtained following the protocol reported earlier for other
pyrethroids (14).
Enzyme Immunoassay. The method was identical to that reported
by Shan et al. (12) with the following exceptions. The wash buffer
was 0.05% Tween 20 in distilled water. The incubation step with
inhibitor was 30 min, and the incubation step with goat anti-rabbit-
IgG-HRP conjugate was 45 min.
Antibody Characterization and Assay Optimization. Antibodies
and antigens were screened in a two-dimensional titration for the best
dilution of coating antigen and antiserum. Then the competitive
inhibition curves were measured for different antibody and antigen
combinations, and the one with the lowest I50 was selected for further
assay development.
The effects of solvents were tested by dissolving the analyte in PBS
buffers containing various proportions of solvent and incubating these
with antibody in PBST on the coated plate. We tested methanol (0,
10, 20,40, 60, and 80%) and DMSO (0, 10, 20, and 40%) in this fashion.
In the experiment to evaluate pH effects, both the analyte and
antiserum were dissolved in PBS buffer at the specified pH and added
to a coated plate. pH values of 5, 6, 7, and 8 were tested in this
incubation step with all other parameters of the assay fixed. Ionic
strength effects were determined in the same manner as previously
mentioned except that, instead of pH, PBS concentration was varied.
PBS concentrations of normal strength (1×), 2×, 4×, and 8× were
tested. Deltamethrin standards were prepared in PBS buffer containing
0, 10, 20, 40, 60, and 80% (v/v) methanol or 0, 10, 20, and 40% (v/v)
DMSO to determine the effects of solvent.
To develop a sensitive and specific deltamethrin immunoas-
say, immunogen hapten design is important. First, because the
alcohol portion of deltamethrin, the phenoxybenzyl group, is
common in many commercial pyrethroids and its acid portion
is relatively unique, our strategy for designing the immunizing
hapten was to link carrier protein through the aromatic end to
improve antibody specificity. Second, a highly lipophilic
pyrethroid conjugated on the protein surface may fold into the
hydrophobic interior of the protein and, thus, result in a low-
affinity antibody. Our previous studies on pyrethroid and dioxin
immunoassays suggested that a short or rigid handle (side chain)
could prevent or minimize such folding (14, 28). Therefore, two
haptens containing the whole or partial deltamethrin molecule
with a short handle on aromatic rings (compounds 3 and 6)
were chosen for immunization. In compound 6, the distal phenyl
group of deltamethrin was eliminated and a carboxylic acid was
directly linked to first aromatic ring.
The deltamethrin haptens 3 and 6 were prepared via acylation
of the cyanohydrins of 3-(4-nitrophenoxy)benzaldehyde and
benzyl 2-(3-formylphenoxy)acetate with the acid chloride of 1R-
cis-deltamethric acid to give intermediates 2 and 5. A small
sample of each was separated by radial chromatography into
the two isomers in order to obtain definitive confirming NMR
spectra. Reduction of the nitro ester 2 with stannous chloride
gave the amino-substituted hapten 3, and cleavage of the benzyl
ester with iodotrimethylsilane gave the hapten 6, each as a
mixture of isomers racemic at the cyanohydrin center.
Cross-Reactivity. The optimized assays were applied to cross-
reactivity studies by using the standard solution of the analytes and
other structurally related compounds. The crosw-reactivity (CR) was
determined by dividing the I50 of the cross-reactant by the I50 of
deltamethrin (assigned as 100%) and multiplying by 100 to obtain a
percent figure.
The coating haptens 10 and 11 were prepared via similar
acylations of the cyanohydrin of 3-(4-nitrophenoxy)benzalde-
hyde as described above using the acid chlorides of (()-cis-
and (()-trans-permethric acid. This introduces an additional
chiral center resulting in two diastereoisomer pairs for each of
the intermediate esters, 8 and 9. A small sample of each ester
was separated by radial chromatography into the two diastere-
oisomer pairs in order to obtain definitive confirming NMR
spectra; however, the respective mixtures were carried through
to the final haptens 10 and 11 by the same reduction reaction
as described for hapten 3. The NMR spectra of the intermediate
esters indicated these pairs to be in a ratio of ∼3:2 in each case.
This ratio allowed tentative spectral assignments to the isomer
pairs of the final haptens. The preparation of haptens 12 and
13 is described in a previous publication (14).
Solid-Phase Extraction. An SPE method used in this study was
the same as a previously reported method for the extraction of
esfenvalerate from water using a C18 column (12). Deltamethrin was
extracted from water using a C18 SPE column (10 cm3/500 mg; part
1211-3027, Varian Samples Preparation Products, Harbor City, CA).
The column was preconditioned with 3.5 mL of methanol and deionized
water before sample application. Water samples (200 mL) were loaded
on the column and eluted with 3-5 mL/min flows. The containers (glass
flask) were washed with deionized water (5-8 × 5 mL), and all washes
were applied onto the column as well. After a thorough drying with
vacuum air, the containers were washed with methanol (3-4 × 1 mL)
and all washes added to the final methanol extract. After the water had
passed through, the column was dried under vacuum for 15 min and
then eluted with 100% methanol (3.5 mL). The mixture of the methanol
extract and washes was evaporated to dryness under a gentle stream of
nitrogen and the residue was redissolved in 1 mL of methanol. An
aliquot diluted with 40% methanol in PBS (1:4) was analyzed by
ELISA.
Screening and Selection of Antisera. The antisera of
terminal bleeds from four rabbits were screened against six
different coating antigens using a two-dimensional titration
method with the coated antigen format. The homologous assay,
in which the same hapten was used in coating antigen and
immunogen, had a higher titer than the heterologous assay.
These results are consistent with a previous study on the
pyrethroid esfenvalerate (12). Ab732 had the highest affinity
RESULTS AND DISCUSSION
Hapten Synthesis. The haptens were synthesized according
to Figure 2. The synthetic routes parallel those described for

5530 J. Agric. Food Chem., Vol. 50, No. 20, 2002
Lee et al.
To identify potential interferences from aqueous environ-
mental samples, the effects of pH and ionic strength on ELISA
performance were evaluated in this study. In system 13/732,
when analyte was dissolved in buffer at different pH values,
no significant effect upon the I50 was detected, indicating that
the assay could effectively detect deltamethrin at pH values
ranging from 5.0 to 8.0. However, ionic strength strongly
influenced ELISA performance. A higher salt concentration in
the assay system resulted in lower optical densities (OD). The
OD values at salt concentrations of 4× and 8× PBS decreased
by 54 and 70%, respectively, from the OD value at a salt
concentration 1× PBS. 2× PBS had the lowest I50 (21.9 µg/L),
followed by 4× PBS (22.3 µg/L) and then 1× PBS (30.0 µg/
L) and 8× PBS (34.7 µg/L). Salt concentrations can affect
antibody binding, and for this assay, a salt concentration slightly
higher than the commonly used PBS improved assay perfor-
mance.
Table 1. Selected Competitive ELISA Screening Data against
Deltamethrina
immunogen
antiserum
coating antigen
I50 (µg/L)
3−THY
729
730
731
3−BSA
3−BSA
12−BSA
13−BSA
11−BSA
10−BSA
3−BSA
6−BSA
12−BSA
13−BSA
11−BSA
10−BSA
3−BSA
6−BSA
2.61 × 106
2.99 × 103
6−THY
119
122
275
148
160
173
203
22.8
42.2
166
732
458
129
a Optimized assay conditions were used. The deltamethrin analyte standards
were prepared in a 40% methanol/PBS solution.
The optimized deltamethrin ELISA used 0.06 µg/mL of
coating antigen 13-BSA, antibody 732 at a dilution of 1/22000,
deltamethrin in 40% methanol/PBS buffer, pH value of 7, and
PBS concentration of 2×. The I50 value of this assay was 17.5
( 3.6 µg/L with a limit of quantitation (LOQ) of 1.1 ( 0.5
µg/L in buffer. The LOQ was estimated as the concentration
that corresponded to the absorbance of the control (zero
concentration of analyte minus 3 times the standard deviation
of the control) (29).
for coating antigen 6-BSA, followed by 13-BSA and then
3-BSA, 10-BSA, 11-BSA, and 12-BSA. Combinations of
coating antigen and antiserum that resulted in high optical
densities (OD > 0.75) were selected for further development.
To identify systems that yielded the highest sensitivity for
deltamethrin, competitive inhibition experiments were conducted
in parallel with optimization of antiserum and coating antigen
concentrations. The reagent concentrations with optical densities
of ∼0.8 and lowest I50 values were chosen as the optimal
combinations. The I50 values for each combination ranged from
22.8 to 2.61 × 106 µg/L (Table 1). The heterologous system
for Ab732 (with cAg 13-BSA) showed the lowest I50 (22.8
µg/L), which was ∼6 times lower than the homologous assay
with cAg 6-BSA. In this study, only the heterologous system
of Ab732 (1/22000) and cAg 13-BSA (0.06 µg/mL) was used
for further assay development and optimization.
Cross-Reactivities. The optimized ELISA system (Ab732/
13-BSA) had the highest CR with cypermethrin (37.3%)
followed by permethrin (9.5%), cyfluthrin (8.5%), phenothrin
(6.8%), and bioresmethrin (2.6%). Because the antibodies were
made to a hapten attached to the protein through the phenoxy-
benzyl group, it was expected that the ability of the antibody
to distinguish among pyrethroids would be less with substitu-
tions in the alcohol portion of the pyrethroid molecule, but still
quite good in the acid portion of the pyrethroid molecule. The
antibody recognizes substitutions on the ethenyl as Br > Cl >
CH3 as demonstrated by decreasing CR for deltamethrin versus
cypermethrin and permethrin versus phenothrin showing the
predicted high selectivity. Because esfenvalerate, fenvalerate,
and fluvalinate do not cross-react (at 5 µg/mL), it is clear that
a cyclopropane ring is important to binding. However, this
crosw-reactivity decreases with the loss of the R-cyano group
(cypermethrin vs permethrin), showing that both the cyclopro-
pane ring and the presence of the R-cyano group are important
determinants to binding. In addition, the antibody is less
selective in the alcohol portion of the molecule as bioresmethrin
can also cross-react.
It is also interesting to note that for one rabbit (731), the I50
values are all similar, indicating that the relationship between
the affinity for the coating antigen and the affinity for delta-
methrin is similar in all cases, regardless of the structure of the
coating antigen. This antiserum may be useful in a more general
pyrethroid screen.
Optimization. For solvent optimization, we tested methanol
as it is commonly used as the eluant in SPE and DMSO because
it is a common cosolvent used in immunoassay to improve
analyte solubility. As observed in our previous studies for
esfenvalerate and permethrin immunoassays (12, 14), MeOH
and DMSO significantly influence the deltamethrin assay
sensitivity and absorbance. The I50 value of the assay varied
depending upon the different concentration of the cosolvent
MeOH or DMSO. The lowest I50 was found at 40% MeOH
(22.3 µg/L), which is ∼25 times lower than that at 10% MeOH
(501 µg/L) and 2 times lower than that at 10% DMSO (42.3
µg/L). The maximum absorbance was enhanced with higher
methanol concentration up to 40% MeOH but was suppressed
in a dose-dependent fashion with DMSO as a cosolvent. A high
concentration of cosolvent facilitates the distribution of hydro-
phobic deltamethrin in the reaction solution. However, very high
levels of organic cosolvents will affect and retard the interaction
of antibody and antigen and may denature protein reagents.
Therefore, a proper concentration of cosolvent is important for
the performance of antibody and assay sensitivity. On the basis
of the I50 values and the ratios of maximum and minimum
absorbances for the deltamethrin standard curve, an MeOH
concentration of 40% was selected for subsequent experiments.
Although the CT of bioresmethrin (1R-trans) is low, it is
interesting that the trans-bioresmethrin isomer shows stronger
CR than the cis-/trans-resmethrin mixture. This is in contrast
to the data from Table 1 that indicate that the antibody binding
is directed more toward the 1R-cis configuration. For example,
deltamethrin, which has a 1R-cis configuration, can inhibit the
assay more when the coating antigen is 1R-trans (haptens 12
vs 13 and 10 vs 11, Table 1).
Among those pyrethroids that show high cross-reactivity,
cypermethrin and cyfluthrin are actively used in the agricultural
field (30). Therefore, cypermethrin will probably be an important
interference when these assays are used for real world sample
measurement. This problem can be addressed by (1) using the
assay as a screening tool, knowing that other pyrethroids were
used in the area to be examined; (2) separating the pyrethroids
chromatographically prior to analysis (31, 32); or (3) utilizing
multiple immunoassays of varying selectivity for pyrethroids

Deltamethrin Immunoassay
J. Agric. Food Chem., Vol. 50, No. 20, 2002 5531
performance liquid chromatography-mass spectrometry; I50,
concentration of analyte giving 50% inhibition; LOQ, limit of
quantitation; NHS, N-hydroxysuccinimide; NMR, nuclear mag-
netic resonance; OD, optical density; PBS, phosphate-buffered
saline; PBST, phosphate-buffered saline with 0.05% of Tween
20; SD, standard deviation; SPE, solid-phase extraction; TLC,
thin-layer chromatography; TMB, tetramethylbenzidine; Thyr,
thyroglobulin.
ACKNOWLEDGMENT
We thank Axel Ehmann (Mass Search, Inc., Modesto, CA) for
mass spectral determinations and the Department of Chemistry,
University of California at Davis, for the use of their NMR
instrument.
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Figure 3. Relationship between deltamethrin spiked into water and
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Received for review July 11, 2002. Accepted July 15, 2002. This work
was supported by the Postdoctoral Fellowship Program of Korea
Science and Engineering Foundation (KOSEF); NIEHS Center P30
ES05707; EPA Center CR819658; California State Water Resources
Control Board Agreement 0-079-250-0; and NATO Collaborative
Linkage Grant 977133. This project was also supported by Grant 5
P42 ES04699-16 from the National Institute of Environmental Health
Sciences, NIH, with funding provided by EPA. Its contents are solely
the responsibility of the authors and do not necessarily represent the
official views of the NIEHS, NIH, or EPA.
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