Development of an immunoassay for the pyrethroid insecticide esfenvalerate

Article abstract of DOI:10.1021/jf981210m

A competitive enzyme-linked immunosorbent assay was developed for the detection of the pyrethroid insecticide esfenvalerate. Two haptens containing amine or propanoic acid groups on the terminal aromatic ring of the fenvalerate molecule were synthesized and coupled to carrier proteins as immunogens. Five antisera were produced and screened against eight different coating antigens. The assay that had the least interference and was the most sensitive for esfenvalerate was optimized and characterized. The I₅₀ for esfenvalerate was 30 ± 6.2 μg/L, and the lower detection limit (LDL) was 3.0 2+ 1.8 μg/L. The assay was very selective. Other pyrethroid analogues and esfenvalerate metabolites tested did not cross-react significantly in this assay. To increase the sensitivity of the overall method, a C₁₈ sorbent-based solid-phase extraction (SPE) was used for water matrix. With this SPE step, the LDL of the overall method for esfenvalerate was 0.1 μg/L in water samples.

Full text of DOI:10.1021/jf981210m

J. Agric. Food Chem. 1999, 47, 2145−2155
2145
Develop m en t of a n Im m u n oa ssa y for th e P yr eth r oid In secticid e
Esfen va ler a te
Guomin Shan,† Donald W. Stoutamire,† Ingrid Wengatz,‡ Shirley J . Gee,† and
Bruce D. Hammock*,†
Department of Entomology, University of California, Davis, California 95616, and EPA-NERL/Human
Exposure Research Branch, P.O. Box 93478, Las Vegas, Nevada 89193-3478
A competitive enzyme-linked immunosorbent assay was developed for the detection of the pyrethroid
insecticide esfenvalerate. Two haptens containing amine or propanoic acid groups on the terminal
aromatic ring of the fenvalerate molecule were synthesized and coupled to carrier proteins as
immunogens. Five antisera were produced and screened against eight different coating antigens.
The assay that had the least interference and was the most sensitive for esfenvalerate was optimized
and characterized. The I50 for esfenvalerate was 30 ( 6.2 µg/L, and the lower detection limit (LDL)
was 3.0 ( 1.8 µg/L. The assay was very selective. Other pyrethroid analogues and esfenvalerate
metabolites tested did not cross-react significantly in this assay. To increase the sensitivity of the
overall method, a C18 sorbent-based solid-phase extraction (SPE) was used for water matrix. With
this SPE step, the LDL of the overall method for esfenvalerate was 0.1 µg/L in water samples.
Keyw or d s: Immunoassay; esfenvalerate; pyrethroids; solid-phase extraction; residue analysis
INTRODUCTION
Esfenvalerate (Figure 1) is a synthetic pyrethroid
widely used for the control of many common pests on
agricultural crops, such as apples, peaches, cotton, and
almonds (Meister, 1996). Due to its excellent insecticidal
F igu r e 1. Structure of esfenvalerate.
properties and low mammalian toxicity (Ecobichon,
trace levels (Hammock and Mumma, 1980; Gee et al.,
1988; Hammock et al., 1990). A number of enzyme-
linked immnosorbert assay (ELISA) methods have been
reported for the detection of synthetic pyrethroids. They
include allethrin (Pullen and Hock, 1995), s-bioallethrin
(1R,3R,4′S-allethrin) (Wing et al., 1978), bioresmethrin
(Hill et al., 1993), deltamethrin (Queffelec et al., 1998;
Lee et al., 1998), fenpropathrin (Wengatz et al., 1998),
and permethrin (Stanker et al., 1989; Skerritt et al.,
1992; Bonwick et al., 1994). To our knowledge no study
has been reported on the development of immunoassays
for esfenvalerate. In this paper, the development of an
ELISA for esfenvalerate and the evaluation of the
assay’s performance in water matrices are described.
1996), the use of this compound has increased rapidly
during the past 15 years. In 1994, >331500 lb of the
active ingredient of esfenvalerate was applied on various
crops in the United States (Gianessi and Anderson,
1995). However, esfenvalerate is extremely toxic to
many aquatic animals (Tomlin, 1997), such as fish, and
amphibious and aquatic invertebrates, which may be
exposed to field runoff or drift from aerial and ground-
based spraying. Numerous studies have shown that
esfenvalerate has detrimental effects on aquatic species
by reduction or elimination of many crustaceans, chi-
ronomids, juvenile bluegills, and larval cyprinids at
exposure levels of 1 ppb (Lozano et al., 1992; Tanner
and Knuth, 1996). Thus, a sensitive, selective, and rapid
method for monitoring residue levels of esfenvalerate
in aquatic ecosystems is desirable.
MATERIALS AND METHODS
Current analytical methods for esfenvalerate rely
upon multistep sample cleanup procedures in conjunc-
tion with high-performance liquid chromatography
(HPLC) (Wells et al., 1994) or gas-liquid chromatog-
raphy (GLC) with either electron capture detection
(Boccelli et al., 1993; Wells et al., 1994) or electrolytic
conductivity detection (Hengel et al., 1997). Such meth-
ods are relatively expensive and skill intensive. An
immunoassay would provide a fast, sensitive, and
selective method for the detection of this pesticide at
Rea gen ts. Cyfluthrin, cypermethrin, deltamethrin, fenval-
erate, fluvalinate, permethrin, phenothrin, and resmethrin
standards (Figure 2) were obtained from Riedel de Haen
(Seelze, Germany). 14C-Labeled fenvalerate (14.9 mCi/mmol)
was provided by Shell Development Co. (Modesto, CA). Race-
mic 4-chloro-R-(1-methylethyl)benzeneacetic acid was gener-
ously supplied by E. I. duPont de Nemours & Co. (Wilmington,
DE). Organic starting materials for hapten synthesis were
obtained from Aldrich Chemical Co. (Milwaukee, WI) and
Fisher Scientific (Pittsburgh, PA). Thin-layer chromatography
(TLC) utilized 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 40 µm
average particle size Baker silica gel using the indicated
solvents, where the f notation denotes a stepwise concentra-
tion gradient.
* Author to whom correspondence should be addressed
[telephone (530) 752-7519; fax (530) 752-1537; e-mail
bdhammock@ucdavis.edu].
† University of California.
‡ EPA-NERL.
10.1021/jf981210m CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/30/1999

2146 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Shan et al.
F igu r e 2. Structures of other pyrethroids used in this study.
The coupling reagents were purchased from Aldrich. Goat
anti-rabbit (GAR) immunoglobulin conjugated to horseradish
peroxidase (HRP), bovine serum albumin (BSA), ovalbumin
(OVA), hemocyanin from Limulus polyphemus (LPH), Tween
20, and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased
from Sigma Chemical Co. (St. Louis, MO).
methylbenzylamine (45.8 g, 0.3 mmol) was added over 15 min.
The mixture was allowed to cool over 1.25 h, then filtered, and
washed with solvent mix. The solid was slurried in methanol
(400 mL), refluxed for 1 h, cooled, and filtered to give 85 g of
a white solid, which was immediately recrystallized from hot
methanol (2.5 L) to give 55 and 22 g in two crops. The latter
was again recrystallized from methanol to give 20 g of product.
The two crops were combined and treated with 3 N HCl, and
the free acid was extracted into diethyl ether. The ether extract
was washed with dilute acid and water and stripped. Recrys-
tallization of the residual solid from hexane (100 mL) provided
46.7 g of (S)-acid, mp 103.5-105.2 °C, [R]2D4 48.1 (c 1.61,
CHCl3). The literature (Miyakado et al., 1975) gives mp 104-5
°C, [R]2D4 46.8 (c 1-2, CHCl3). The remaining amine salt
concentrated in the (R)-acid fraction was treated with HCl to
recover the free acid. Treatment in the same way with (R)-
(+)-R-methylbenzylamine and workup as above led to 46 g of
the (R)-acid as a white solid: mp 101-105.5 °C, [R]2D4-47.5 (c
1.49, CHCl3).
The optical purity of each acid was determined by treatment
of a 100 mg (0.47 mmol) sample in 0.3 mL of CHCl3 plus 0.5
µL of N,N-dimethylformamide (DMF) with SOCl2 (0.068 mL,
0.93 mmol) at 60 °C for 45 min. Vacuum stripping of the
resulting acid chloride followed by addition of CH2Cl2 (0.5 mL)
to an ice-chilled solution of (S)-(-)-R-methylbenzylamine (0.13
mL, 1.0 mmol) (Aldrich, 99.6% ee) in CH2Cl2 (0.5 mL) gave
the diastereoisomeric amides. GLC analysis (50 f 300 °C at
20 °C/min) gave complete separation of enantiomer pairs; the
(SS + RR)/(SR + RS) ratios for the (S)- and (R)-acids were
98.3:1.7 and 0.8:99.2, respectively. Conversion of the acids to
their respective amides using 1,3-dicyclohexylcarbodiimide
(DCC) gave similar results: 97.7:2.3 and 0.6:99.4, respectively.
The minimum optical purities of the two acids are therefore
approximately 96 and 98% ee, respectively.
Ap p a r a tu s. NMR spectra 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 Uni-Melt apparatus (Thomas Scientific, Swedes-
borogh, NJ ) and are uncorrected. Gas-liquid chromatograms
were determined on an HP 5890 (Hewlett-Packard Corp.,
Avondale, PA) with a 15 m, 0.32 mm i.d., capillary column
filled with a 0.25 µm film of dimethylpolysiloxane containing
5% of methyls substituted by phenyls (J &W Scientific, Folsom,
CA). 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-HS-2F mass spectrometer (VG Analytical, Wythen-
shawe, U.K.), using xenon (8 keV, 1 mA) for ionization and
3-nitrobenzyl alcohol or glycerol as the matrix. Poly(ethylene
glycol) was added to the matrix as a mass calibrant. 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). Two brands of
microplates were used: Nunc-Immuno plates were used for
ELISAs, and Dynatech microtiter plates (Dynatech Labora-
tories, Inc., Chantilly, VA) were used for preparing serial
dilutions.
Ha p ten Syn th esis a n d Ver ifica tion . Syntheses of the
haptens were carried out as outlined in Scheme 1. All reactions
were straightforward, and yields were moderate to good.
Analytical data supporting the structures are provided. Ra-
cemic 4-chloro-R-(l-methylethyl)benzeneacetic acid (fenvalerate
acid) was resolved via the (R)- and (S)-R-methylbenzylamine
salts as described.
Resolu tion of 4-Ch lor o-r-(1-m eth yleth yl)ben zen ea ce-
tic Acid (F en va ler a te Acid ). The racemic acid (128 g, 0.6
mol) in toluene (218 mL) and methanol (320 mL) was stirred
and heated to reflux, and, with heating removed; (S)-(-)-R-
F en va ler a te Isom er Sta n d a r d s. 3-Phenoxybenzaldehyde
(2.38 g, 12 mmol) and potassium cyanide (1.17 g, 18 mmol) in
a mixture of 7 mL of tetrahydrofuran (THF) and 0.5 mL of
water was stirred with ice cooling, and concentrated HCl (1.3
mL, 16 mmol) was added all at once. After a short time, the
mixture became basic and a mild exotherm accompanied the
formation of the cyanohydrin. The mixture was allowed to
warm to room temperature with stirring over 30 min. The
mixture was diluted with CH2Cl2 and water, acidified with 3

Esfenvalerate Immunoassay
J. Agric. Food Chem., Vol. 47, No. 5, 1999 2147
Sch em e 1. Syn th esis of Ha p ten s 4, 7, 10, 11, a n d 13
N HCl (caution, HCN released), washed three times with
water, dried briefly over anhydrous MgSO4, and stripped to a
colorless oil. Meanwhile, (S)-fenvalerate acid (2.55 g, 12 mmol)
in 6 mL of CHCl3 was treated with SOCl2 (1.31 mL, 1.8 mmol)
and DMF (0.5 µL) and stirred while heating in an oil bath at
60 °C for 1 h. The mixture was stripped, and 5 mL of hexane
was added and stripped to give the acid chloride as a colorless
oil. This acid chloride in 4 mL of CH2Cl2 was stirred with ice
cooling, and the above cyanohydrin in 4.5 mL of CH2Cl2 was
added all at once followed immediately with 1.2 mL of pyridine.
After an immediate mild exotherm, the mixture was stirred
for 30 min, diluted with water, and acidified with 3 N HCl.
The organic phase was washed with dilute HCl and water,
dried over MgSO4, and stripped to an oil, which was flash
chromatographed on 100 g of silica gel (10 f 60% CH2Cl2 in
hexane). Stripping product fractions briefly to 60 °C under high
vacuum gave 4.68 g (95%) of a mixture of the S,S and S,R
isomers of fenvalerate insecticide. A 1.2 g sample of this
mixture was separated by radial chromatography (2 mm silica
gel, 0.5% THF in hexane) in six 200 mg portions to give nearly
complete separation of diastereoisomers. Each of the isomers
was repassed in three portions for further purification. Sepa-
ration was followed by TLC (10 cm plates), for which five or
more solvent passes (5% THF in hexane) were required to show
complete separation of the two isomers. Stripping the lower
Rf S,S isomer gave 620 mg of oil, which was crystallized from
∼2 mL of MeOH at 0 °C to give 495 mg of white solid: mp
60-61.5 °C, [R]2D4 -11.1 (c 1.5, CHCl3); 1H NMR (CDCl3) δ
0.70 (d, J ) 6.7 Hz, 3 H, CH3), 0.95 (d, J ) 6.5 Hz, 3 H, CH3),
2.30 (m, J ) 6.6, 10.5 Hz, 1 H, Me2CH), 3.22 (d, J ) 10.5 Hz,
1 H, CHC(O)), 6.34 (s, 1 H, CHCN), 6.98-7.40 (m, 13 H, Ar).
A second crop was recrystallized to give 19 mg of additional
product, mp 59-61 °C. Stripping the higher Rf S,R isomer
fractions in the same way gave 645 mg of a colorless viscous
liquid: [R]2D4 -11.2 (c 0.985, CHCl3); H NMR (CDCl3) δ 0.73
1
(d, J ) 6.7 Hz, 3 H, CH3), 1.06 (d, J ) 6.5, 3 H, CH3), 2.33 (m,
J ) 6.6, 10.4 Hz, 1 H, Me2CH), 3.23 [d, J ) 10.7 Hz, 1 H, CHC-
(O)], 6.29 (s, 1 H, CHCN), 6.96-7.38 (m, 13 H, Ar).
A similar reaction and workup sequence starting with (R)-
fenvalerate acid resulted in 441 mg of the R,R isomer [mp 59-
60.5 °C, [R]2D4 12.0 (c 1.15, CHCl3)] and 575 mg of the R,S
isomer as an oil [[R]2D4 12.7 (c 1.72, CHCl3)]. GLC analysis (15
m capillary DB-5 column, 50 f 300 °C at 20 °C/min) of the
four chiral fenvalerate isomers gave the following results: S,S
isomer, >98% under one peak, 1.4% corresponding to S,R/R,S
isomers; S,R isomer, >99% under one peak; R,R isomer, >98%
under one peak, 1.4% corresponding to R,S/S,R isomers; R,S
isomer, >99% under one peak.
Cya n o[3-(4-n itr op h en oxy)p h en yl]m eth yl (S)-4-Ch lor o-
r-(1-m eth yleth yl)ben zen eacetate (4-Nitr ofen valer ate) (3).
The cyanohydrin of 3-(4-nitrophenoxy)benzaldehyde (2) (Loewe
and Urbanietz, 1967) (3 g, 12.3 mmol) was prepared as
described in a previous publication (Wengatz et al., 1998).
Meanwhile, the acid chloride of (S)-fenvalerate acid (2.62 g,
12.3 mmol) was prepared as described above. The above
cyanohydrin in 10 mL of CH2Cl2 was stirred with ice cooling
as the acid chloride in 10 mL of CH2Cl2 was added followed
immediately with pyridine (1.25 mL, 15.4 mmol). After an
initial mild exotherm, the mixture was stirred at room
temperature for 20 min. The resulting mixture was washed
with 3 N HCl solution and water, and the organic phase was

2148 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Shan et al.
dried (MgSO4) and stripped to a yellow gum. Flash chroma-
tography on 85 g of silica gel (hexane f CH2Cl2) and high-
vacuum stripping gave the pure ester as a mixture of the two
diastereoisomers, 5.08 g (89%): TLC Rf 0.55 (CH2Cl2). Separa-
tion of a ∼200 mg sample by radial chromatography (2 mm
silica gel, 5% THF in hexane) gave complete separation in two
CH3), 2.3 (m, J ) 6.5, 10.4 Hz, 1 H, Me2CH), 2.7 (t, J ) 7.6
Hz, 2 H, CH2), 3.0 (t, J ) 7.6 Hz, 2 H, CH2), 3.23 [d, J ) 10.4
Hz, 1 H, CHC(O)], 5.12 (s, 2 H, CH2Ar), 6.28 (s, 1 H, CHCN),
6.86-7.4 (m, 17 H, Ar); S,S isomer δ 0.72 (d, J ) 6.9 Hz, 3 H,
CH3), 0.94 (d, J ) 6.5 Hz, 3 H, CH3), 2.3 (m, J ) 6.5, 10.4 Hz,
1 H, Me2CH), 2.7 (t, J ) 7.6 Hz, 2 H, CH2), 3.0 (t, J ) 7.6 Hz,
2 H, CH2), 3.22 [d, J ) 10.4 Hz, 1 H, CHC(O)], 5.12 (s, 2 H,
CH2Ar), 6.33 (s, 1 H, CHCN), 6.86-7.4 (m, 17 H, Ar).
passes: 100 mg of the higher Rf S,R isomer [[R]21 -14.1 (c
D
1
1.13, CHCl3); H NMR δ 0.73 (d, J ) 6.7 Hz, 3 H, CH3), 1.03
(d, J ) 6.5 Hz, 3 H, CH3), 2.34 (m, J ) 6.6, 10.5 Hz, 1 H, Me2-
CH), 3.24 [d, J ) 10.5 Hz, 1 H, CHC(O)], 6.37 (s, 1 H, CHCN),
4-[3-[Cya n o[(S)-2-(4-ch lor op h en yl)-3-m eth yl-1-oxobu -
tan oxy]m eth yl]]ph en oxy]ben zen epr opan oic Acid (7). The
ester 6 as the diastereoisomer pair (0.97 g, 1.67 mmol) in 2
mL of CH2Cl2 was treated under N2 with 25 µL of BSTFA
followed by iodotrimethylsilane (0.249 mL, 1.75 mmol). After
3 h, the mixture was treated with 1 mL of water. The organic
phase was then immediately flash chromatographed on 20 g
of silica gel (CH2Cl2 f EtOAc) to give 0.81 g (99%) of the acid
as a pale yellow gum after high-vacuum stripping, which was
one spot by TLC: Rf 0.35 (EtOAc). 1H NMR (CDCl3) showed a
1:1 mixture of two diastereoisomers. Comparison to spectra
of fenvalerate and nitrofenvalerate isomers allowed tentative
assignment of the two spectra as follows: S,R isomer δ 0.70
(d, J ) 6.86 Hz, 3 H, CH3), 1.05 (d, J ) 6.5 Hz, 3 H, CH3), 2.3
(m, J ) 6.5, 10.2 Hz, 1 H, Me2CH), 2.69 (t, J ) 7.6 Hz, 2 H,
CH2), 2.96 (t, J ) 7.7 Hz, 2 H, CH2), 3.23 [d, J ) 10.5 Hz, 1 H,
CHC(O)], 6.29 (s, 1H, CHCN), 6.9-7.39 (m, 12 H, Ar); S,S
isomer δ 0.72 (d, J ) 6.86 Hz, 3 H, CH3), 0.95 (d, J ) 6.5 Hz,
3 H, CH3), 2.3 (m, J ) 6.5, 10.2 Hz, 1 H, Me2CH), 2.69 (t, J )
7.6 Hz, 2 H, CH2), 2.96 (t, J ) 7.7 Hz, 2 H, CH2), 3.22 [d, J )
10.5 Hz, 1 H, CHC(O)], 6.34 (s, 1 H, CHCN), 6.9-7.39 (m, 12
7.0-8.3 (m, 12 H, Ar)] and 89 mg of the lower Rf S,S isomer
1
[[R]21 -7.3 (c 1.16, CHCl3); H NMR (CDCl3) δ 0.72 (d, J )
D
6.7 Hz, 3 H, CH3), 0.96 (d, J ) 6.5 Hz, 3 H, CH3), 2.30 (m, J
) 6.6, 10.5 Hz, 1 H, Me2CH), 3.24 [d, J ) 10.5 Hz, 1 H, CHC-
(O)], 6.40 (s, 1 H, CHCN), 7.0-8.3 (m, 12 H, Ar)]. The absolute
configuration of the latter was determined as follows: A 15
mg sample was reduced in 0.25 mL of EtOH with 44 mg of
SnCl2‚2H2O (30 min/70 °C) to give 10.5 mg of the correspond-
ing aniline after chromatography: 1H NMR (CDCl3) δ 0.7 (d,
J ) 6.5 Hz, 3 H, CH3), 0.95 (d, J ) 6.5 Hz, 3 H, CH3), 2.30 (m,
1 H, Me2CH), 3.21 [d, J ) 10.4 Hz, 1 H, CHC(O)], 3.64 (b, 2 H,
NH2), 6.30 (s, 1 H, CHCN), 6.68-7.36 (m, 12 H, Ar). This was
dissolved in 0.3 mL of H2O plus 0.22 mL of 3 N HCl plus 0.2
mL of EtOH, chilled in ice, and treated with 1 equiv of NaNO2
solution followed by 0.15 mL of 50% H3PO2. Workup and
purification by TLC gave an oil, which crystallized immediately
on seeding with, and had an identical TLC to, (S,S)-fenvalerate
[five solvent passes (5% THF in hexane) gave complete
separation of the two diastereoisomers].
+
H, Ar); FAB-HRMS m/z calcd for [M + H] ) C28H27ClNO5
Cyan o[3-(4-am in oph en oxy)ph en yl]m eth yl (S)-4-Ch lor o-
r-(1-m et h ylet h yl)b en zen ea cet a t e (4-Am in oesfen va ler -
a te) (4). Stannous chloride hydrate (226 mg, 1 mmol) was
added to a stirred solution of (S,RS)-nitrofenvalerate 3 (93 mg,
0.2 mmol) in 0.75 mL of EtOH, and the mixture was heated
under N2 at 70-75 °C for 30 min. The cooled mixture was
treated with 0.4 g of filter aid and diluted with water and
ether. NaHCO3 (168 mg, 2 mmol) was added in portions and
stirred until CO2 evolution ceased. The mixture was filtered,
and solids were extracted with ether. Combined ether extracts
were stripped, and the residue was chromatographed on 5 g
of silica gel (50% CH2Cl2/hexane f CH2Cl2) to recover 5 mg of
starting material and 53.9 mg (62%) of the aniline as a
colorless viscous gum: TLC Rf 0.3 (CH2Cl2). The TLC spot
rapidly darkened on exposure to light. A similar reduction of
the S,S isomer (186 mg, 0.4 mmol) gave, after chromatography,
123 mg (70%) as a viscous colorless oil, which had a TLC Rf
identical to that of the S,R/S isomer mix obtained above: 1H
NMR (CDCl3) δ 0.70 (d, J ) 6.7 Hz, 3 H, CH3), 0.95 (d, J ) 6.5
Hz, 3 H, CH3), 2.30 (m, 1 H, Me2CH), 3.21 [d, J ) 10.4 Hz, 1
H, CHC(O)], 3.64 (b, 2 H, NH2), 6.30 (s, 1 H, CHCN), 6.68-
7.36 (m, 12 H, Ar); 13C NMR (CDCl3) δ 19.9, 21.0, 31.9, 58.6,
62.7, 115.5, 115.9, 116.2, 118.7, 120.8, 121.2, 128.7, 129.7,
130.3, 133.0, 133.5, 135.1, 143.3, 147.4, 159.6, 171.3; FAB-
HRMS m/z calcd for [M+] ) C25H23ClN2O3 434.1396, obsd
434.1384.
492.1552, obsd 492.1577.
Ben zyl 3-[Cya n o[(S)-2-(4-ch lor op h en yl)-3-m et h yl-1-
oxobu ta n oxy]m eth yl]p h en oxya ceta te (8). Benzyl 2-(3-
formylphenoxy)acetate (2.0 g, 7.4 mmol) was converted to the
cyanohydrin as described in a previous paper (Wengatz et al.,
1998). (S)-4-Chloro-R-(1-methylethyl)benzeneacetyl chloride
was prepared from the corresponding (S)-acid (1.57 g, 7.4
mmol) as described above. The cyanohydrin in 2.5 mL of CH2-
Cl2 was stirred with ice cooling, and the acid chloride in 3 mL
of CH2Cl2 was added all at once followed after 5 min by
pyridine (0.75 mL, 9.2 mmol). The mixture was stirred at room
temperature for 30 min, then washed with water and a
saturated NaCl solution, dried (MgSO4), and stripped to a pale
yellow oil. Flash chromatography on 70 g of silica gel (5 f
90%) CH2Cl2 in hexane and high-vacuum stripping of product
fractions gave 3.25 g (90%) as a mixture of diastereoisomers.
A sample of this mixture (1.23 g) was separated by radial
chromatography (2 mm silica gel, 5% THF in hexane) in ∼200
mg batches to give nearly complete separation of isomers. For
final purification, the higher Rf isomer was repassed with the
same chromatographic condition to recover 613 mg of colorless
viscous oil after high-vacuum stripping: [R]2D6 -5.42 (c 1.62,
CHCl3); 1H NMR (CDCl3) δ 0.73 (d, J ) 6.6 Hz, 3 H, CH3),
1.06 (d, J ) 6.3 Hz, 3 H, CH3), 2.33 (m, J ) 6.7, 10.3 Hz, 1 H,
Me2CH), 3.23 [d, J ) 10.5 Hz, 1 H, CHC(O)], 4.6 [s, 2 H,
OCH2C(O)], 5.24 (s, 2 H, CH2Ph), 6.28 (s, 1 H, CHCN), 6.83-
7.42 (m, 13 H, Ar). The lower Rf isomer was further purified
in the same way to recover 586 mg of colorless viscous oil:
[R]2D6 -8.26 (c 1.84, CHCl3); 1H NMR (CDCl3) δ 0.70 (d, J )
6.7 Hz, 3 H, CH3), 0.95 (d, J ) 6.5 Hz, 3 H, CH3), 2.30 (m, J
) 6.4, 10.4 Hz, 1 H, Me2CH), 3.22 [d, J ) 10.5 Hz, 1 H, CHC-
(O)], 4.66 [s, 2 H, OCH2C(O)], 5.24 (s, 2 H, CH2Ph), 6.32 (s, 1
H, CHCN), 6.94-7.39 (m, 13 H, Ar). Comparison with the very
consistent NMR shifts observed with the fenvalerate isomers
and the nitro- and aminofenvalerate above allowed tentative
assignment of the S,R and S,S configurations to the higher
and lower Rf isomers, respectively.
Ben zyl 4-[3-[Cya n o[(S)-2-(4-ch lor op h en yl)-3-m eth yl-1-
oxobu ta n oxy]m eth yl]]p h en oxy]ben zen ep r op a n oa te (6).
Benzyl 4-(3-formylphenoxy)benzenepropanoate (3.6 g, 10 mmol)
was converted to the cyanohydrin as described in a previous
paper (Wengatz et al., 1998). (S)-4-Chloro-R-(1-methylethyl)-
benzeneacetyl chloride was prepared from the corresponding
(S)-acid (2.13 g, 10 mmol) as described above. The acid chloride
in 7 mL of CH2Cl2 was stirred with ice cooling, and the
cyanohydrin in 7 mL of CH2Cl2 was added all at once followed
immediately by pyridine (1.01 mL, 12.5 mmol). The mixture
was stirred at ambient temperature for 30 min and then
washed with water, 3 N HCl, and water, dried (MgSO4), and
stripped to light brown oil. Flash chromatography on 80 g of
silica gel (50% hexane/CH2Cl2 f ether) gave 4.96 g (85%) of
product as a mixture of two diastereoisomers. 1H NMR (CDCl3)
indicated a 1:1 mixture of diastereoisomers. Comparison to
spectra of fenvalerate and nitrofenvalerate isomers allowed
tentative assignment of the two spectra as follows: S,R isomer
δ 0.70 (d, J ) 6.9 Hz, 3 H, CH3), 1.05 (d, J ) 6.5 Hz, 3 H,
Ben zyl 6-[3-[Cya n o[(S)-2-(4-ch lor op h en yl)-3-m eth yl-1-
oxobu ta n oxy]m eth yl]p h en oxy]h exa n oa te (9). Benzyl 6-(3-
formylphenoxy)hexanoate (1.75 g, 5.36 mmol) was converted
to the cyanohydrin as described in a previous paper (Wengatz
et al., 1998). (S)-4-Chloro-R-(1-methylethyl)benzeneacetyl chlo-
ride was prepared from the corresponding (S)-acid (1.14 g, 5.36
mmol) as described above. These two materials were reacted

Esfenvalerate Immunoassay
J. Agric. Food Chem., Vol. 47, No. 5, 1999 2149
using the same procedure as described for the ester 8 above.
The crude stripped product was flash chromatographed on 60
g of silica gel (10% CH2Cl2/hexane f CH2Cl2) to give 7% of
the starting aldehyde and 2.66 g (90%) of the ester 9, a mixture
of two diastereoisomers, as a colorless viscous oil. A 1.3 g
sample of this mixture was separated by radial chromatogra-
phy (2 mm silica gel, 5% THF in hexane) in ∼200 mg portions
with recycle of overlap fractions into the following portion.
Each of the isomers was repassed in two portions for further
purification. Separation was followed by TLC (10 cm plates)
with which five or more solvent passes (5% THF in hexane)
were required to show complete separation of the two isomers.
High-vacuum stripping of the higher Rf S,R isomer gave 637
mg of colorless oil: [R]2D4 -7.76 (c 1.12, CHCl3); 1H NMR
(CDCl3) δ 0.72 (d, J ) 6.6 Hz, 3 H, CH3), 1.07 (d, J ) 6.4 Hz,
3 H, CH3), 1.49 (m, 2 H, CH2), 1.75 (m, 4 H, 2 CH2), 2.35 (m,
1 H, Me2CH), 2.4 (t, J ) 7.4 Hz, 2 H, CH2), 3.23 [d, J ) 10.4
Hz, 1 H, CHC(O)], 3.84 (t, J ) 6.18 Hz, 2 H, CH2), 5.12 (s, 2
H, C6H5CH2), 6.3 (s, 1 H, CHCN), 6.78-7.35 (m, 13 H, Ar).
Similar stripping of the lower Rf S,S isomer gave 623 mg:
[R]2D4 -13.8 (c 1.31, CHCl3); 1H NMR (CDCl3) δ 0.70 (d, J )
6.7 Hz, 3 H, CH3), 0.97 (d, J ) 6.5 Hz, 3 H, CH3), 1.51 (m, 2 H,
CH2), 1.77 (m, 4 H, 2 CH2), 2.31 (m, 1 H, Me2CH), 2.41 (t, J )
7.4 Hz, 2 H, CH2), 3.22 [d, 1 H, CHC(O)], 3.91 (t, J ) 6.3 Hz,
2 H, CH2), 5.12 (s, 2 H, C6H5CH2), 6.34 (s, 1 H, CHCN), 6.91-
7.36 (m, 13 H, Ar).
detectable. The mixture was diluted with CH2Cl2 and acidified
(3 N HCl) to precipitate a white solid. After filtration, the
organic phase precipitated additional solid material. Tritura-
tion of the filtrate oil with ether gave additional solid. All three
crops (∼2.2 g) were a mixture of two components by TLC: Rf
) 0.26 and 0.45 (3% HOAc in EtOAc); Rf ) 0.53 and 0.65 (3%
HOAc in acetone). These were separated with some difficulty
by column chromatography on 215 g of silica gel [(3% HOAc)
CH2Cl2 f EtOAc]. Recrystallization of the higher Rf product
(THF diluted with n-butyl chloride) recovered 1.1 g (35%) of
12: mp 187.5-188 °C; [R]2D4 -52.4 (c 1.06, EtOH); 1H NMR
(DMSO-d6) δ 0.51 (d, J ) 6.6 Hz, 3 H, CH3), 0.57 (d, J ) 6.4
Hz, 3 H, CH3), 2.05 (dquin, J ) 6.5, 10.8 Hz, 1 H, Me2CH),
2.96 (d, J ) 10.7 Hz, 1 H, Me2CHCH), 3.0 (dd, J ) 10.7, 13.7
Hz, 1 H, HCHAr), 3.22 (dd, J ) 4.6, 13.6 Hz, 1 H, HCHAr),
4.50 [m, 1 H, NCHC(O)], 7.26 (d, J ) 8.6 Hz, 2 H, ArCl), 7.33
(d, J ) 8.6 Hz, 2 H, ArCl), 7.42 (d, J ) 8.7 Hz, 2 H, ArNO2),
8.07 (d, J ) 8.7 Hz, 2 H, ArNO2), 8.43 (d, J ) 8.4 Hz, 1 H,
NH), 12.7 (bs, 1 H, COOH); FAB-MS m/z calcd for [M + H]+
20H22ClN2O5 405.12, obsd 405. Recrystallization of the lower
)
C
Rf product from the same solvent mix gave 0.4 of white solid:
mp 244-247 °C; [R]2D1 -20.1 (c 1.03, EtOH), which was
indicated by NMR and MS to be the acyl-4-nitrophenylalanyl-
4-nitrophenylalanine; FAB-MS m/z calcd for [M + H]+
)
C
29H30ClN4O8 597.195, obsd 597.
N -[(S )-4-C h lo r o -2-(1-m e t h y le t h y l)b e n ze n e a c e t y l]-
4-a m in o-L-p h en yla la n in e (13). A 0.75 g (1.85 mmol) sample
of 12 and 25 mg of 10% Pd on carbon in 20 mL of ethanol was
stirred under an atmosphere of hydrogen for 2.5 h. A TLC
indicated conversion to a single major product, Rf 0.46 (3%
HOAc in EtOAc), which darkened on exposure to light. A
slightly more polar impurity was assumed to be the dechlori-
nated analogue. This was removed by flash chromatography
on silica gel (3% HOAc in CH2Cl2 f EtOAc) to recover a
colorless gum. This product was crystallized from diethyl ether
to give 0.664 g (72%) of a pale tan solid: mp 105-9 °C (gas);
[R]2D4 5.6 (c 1.04, EtOH), which was shown by NMR to be a 1:1
molecular compound with ether; stripping the NMR sample
and reanalysis removed the ether spectrum; 1H NMR (DMSO-
d6) δ 0.54 (d, J ) 6.6 Hz, 3 H, CH3), 0.70 (d, J ) 6.4 Hz, 3 H,
CH3), 2.07 (dquin, J ) 6.6, 10.7 Hz, 1 H, Me2CH), 2.69 (dd, J
) 9.6, 13.8 Hz, 1 H, CH2), 2.87 (dd, J ) 4.7, 13.8 Hz, 1 H,
CH2), 3.08 (d, J ) 10.6 Hz, 1 H, Me2CHCH), 4.30 (dq, J ) 4.6,
8.5 Hz, 1 H, NCHCOO), 6.51 (d, J ) 8.2 Hz, 2 H, Ar), 6.86 (d,
J ) 8.2, 2 H, Ar), 7.28-7.35 (m, 4 H, Ar), 8.29 (d, J ) 8.2 Hz,
1 H, NHCO); FAB-MS m/z calcd for [M + H]+ ) C20H24ClN2O3
375.04, obsd 375.
3-[Cya n o[(S)-2-(4-ch lor op h en yl)-3-m eth yl-1-oxobu ta n -
oxy]m eth yl]p h en oxya cetic Acid (10). The ester 8 as the
diastereoisomer pair (1.78 g, 3.6 mmol) in 3.5 mL of CH2Cl2
was treated with 30 µL of BSTFA and iodotrimethylsilane
(0.523 mL, 3.68 mmol). After 20 h at ambient temperature,
1.5 mL of methanol was added, and the mixture was washed
with water and saturated NaCl solution, dried (MgSO4), and
stripped. The resulting gum was immediately flash chromato-
graphed on 25 g of silica gel (CH2Cl2 f EtOAc f 3% HOAc in
EtOAc). Fractions containing only product by TLC, Rf 0.4 (CH2-
Cl2), were stripped under high vacuum to give 1.45 g (100%)
of 10 as the diastereoisomer pair. A sample of the S,S isomer
of 10 (450 mg, 0.915 mmol) was similarly cleaved and purified
to give 10 (300 mg) as the pure S,S isomer: 1H NMR (CDCl3)
δ 0.71 (d, J ) 6.7 Hz, 3 H, CH3), 0.96 (d, J ) 6.5 Hz, 3 H,
CH3), 2.31 (m, J ) 6.6, 10.4 Hz, 1 H, Me2CH), 3.23 [d, J )
10.5 Hz, 1 H, CHC(O)], 4.68 [s, 2 H, OCH2C(O)], 6.35 (s, 1 H,
CHCN), 6.75 (b, 1 H, COOH), 6.97-7.39 (m, 8 H, Ar); FAB-
+
MS m/z calcd for [M + H] ) C21H21ClNO5 402.1, obsd 402.
6-[3-[Cya n o[(S )-2-(4-ch lor op h e n yl)-3-m e t h yl-1-oxo-
bu ta n oxy]m eth yl]p h en oxy]h exa n oic Acid (11). The ester
9 as the S,S isomer (400 mg, 0.73 mmol) in 0.75 mL of CH2Cl2
was treated under N2 with BSTFA (30 µL) and iodotrimeth-
ylsilane (0.109 mL, 7.65 mmol). After 12 h, the mixture was
treated with 0.5 mL of methanol, washed three times with
water, and stripped. Flash chromatography on 7 g of silica gel
(CH2Cl2 f 3% HOAc in EtOAc) recovered 12 mg of the starting
ester and 261 mg (78%) of the acid 11 as viscous oil: 1H NMR
(CDCl3) δ 0.71 (d, J ) 6.7 Hz, 3 H, CH3), 0.97 (d, J ) 6.5 Hz,
3 H, CH3), 1.54 (m, 2 H, CH2), 1.73 (m, 2 H, CH2), 1.82 (m, 2
H, CH2), 2.31 (m, J ) 6.5, 10.5 Hz, 1 H, Me2CH), 2.41 (t, J )
7.4 Hz, 2 H, CH2), 3.25 [d, J ) 10.5 Hz, 1 H, CHC(O)], 3.93 (t,
J ) 6.3 Hz, 2 H, CH2), 6.35 (s, 1 H, CHCN), 6.93-7.34 (m, 8
H, Ar); 13C NMR (CDCl3) δ 19.9 (CH3), 21.0 (CH3), 24.3, 25.4,
28.7, 31.9, 33.8, 58.7, 62.9, 67.7, 113.5, 115.6, 116.4, 119.6,
128.7 (2C), 129.7 (2C), 130.2, 132.8, 133.5, 135.2, 159.4, 171.4,
Ha p ten Con ju ga tion . Conjugates were synthesized using
three different methods: water soluble carbodiimide, diazoti-
zation, and activated ester method (Tijssen, 1985; Erlanger,
1973). To obtain immunogens, haptens 4 and 7 were conju-
gated to LPH. Coating antigens were made by coupling
haptens 4, 7, 10, 11, and 13 to BSA and/or OVA.
Con ju ga tes of 4-BSA/LP H a n d 13-BSA/OVA. Hapten
4 or 13 (0.104 mmol) was dissolved in 4 drops of ethanol and
treated with 1 mL of 1 N HCl. The resulting solution was
stirred in an ice bath as 0.5 mL of a 0.201 M solution of sodium
nitrite was injected. Some product appeared as a gel. DMF
(0.4 mL) was added dropwise to give a homogeneous solution,
which was divided into two equal aliquots (each aliquot was
used for one protein). Ninety-eight milligrams of LPH or 101
mg of BSA or 100 mg of OVA was dissolved in 30 mL of 0.2 M
borate buffer (pH 8.8) and 1.5 mL of DMF. Aliquots (∼0.9 mL
each) of the activated hapten solution were added dropwise
to the two stirred protein solutions (hapten 4 for LPH and
BSA; hapten 13 for BSA and OVA). The reaction mixture was
stirred in an ice bath for 45 min and purified by acetone
precipitation as described in Wengatz et al. (1998). The
purified conjugates were suspended in water and stored in
aliquots at -80, -20, and 4 °C.
+
179.7); FAB-HRMS m/z calcd for [M + H] ) C25H29ClNO5
458.1734, obsd 458.175.
N-[(S)-4-Ch lor o-r-(1-m eth yleth yl)ben zen ea cetyl)-4-n i-
tr o-L-p h en yla la n in e (12). (S)-4-Chloro-R-(1-methylethyl)-
benzeneacetyl chloride was prepared from the corresponding
(S)-acid (1.71 g, 8 mmol) as described above. This was added
in portions over 15 min to a vigorously stirred ice-cooled
solution of 4-nitro-L-phenylalanine hydrate (1.75 g, 7.67 mmol)
and 4-(dimethylamino)pyridine (4 mg) in 3.84 mL of 2 N KOH
and 3 mL of water. An additional 3.8 mL of 2 N KOH was
added dropwise over 45 min to maintain a slightly basic pH.
After an additional 2 h, the odor of acid chloride was no longer
Con ju ga tes of 7-BSA/LP H. Morpho-CDI (100 mg, 0.236
mmol) was added to a solution of 55 mg (0.112 mmol) of hapten
7 in 1 mL of DMF. The resulting mixture was diluted with
additional DMF (2 mL) and then diluted dropwise with water

2150 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Shan et al.
almost to the point of oil-out. The solution was divided into
two aliquots. Ninety-eight milligrams of LPH or 102 mg of BSA
was dissolved in 16 mL of cold distilled H2O and the pH was
adjusted to 6.5 with 0.2 M HCl. DMF (0.75 mL) was then
added to each protein solution. Aliquots (2.25 mL) of the
activated hapten solution were added slowly with stirring. The
reaction mixture was stirred for 2 h at room temperature and
purified and stored as described above.
Con ju ga tes of 10 (a n d 11)-BSA/OVA. Hapten (0.05
mmol) was dissolved in 2 mL of dry DMF, and then NHS (18
mg, 0.08 mmol) and DAPEC (13.6 mg, 0.06 mmol) were added.
The reaction mixture was stirred overnight at room temper-
ature and divided into two aliquots. Each aliquot was used
for one protein. Twenty milligrams of BSA or OVA was
dissolved in 3 mL of PBS (pH 7.6). Aliquots of the activated
hapten solutions were added dropwise to the two protein
solutions. The mixture was stirred for 1.5 h at 4 °C and for 7
h at room temperature and then purified using dextran
desalting columns as described in Wengatz et al. (1998) and
stored as described above.
Im m u n iza tion a n d An tiser u m P r ep a r a tion . Esfenval-
erate antisera were obtained following the protocol reported
earlier (Wengatz et al., 1998). Briefly, for each immunogen (4-
LPH, 7-LPH), three New Zealand white rabbits were im-
munized. The antigen solutions (100 µg in PBS) were emul-
sified with Freund’s complete adjuvant (1:1, v/v) and injected
intradermally. After 1 month, the animals were boosted with
an additional 100 µg of immunogen that was emulsified with
Freund’s incomplete adjuvant (1:1, v/v). Booster injections were
given at 3 week intervals. The rabbits were bled 10 days after
each boost. One rabbit (no. 7587) died prior to the final bleed.
The serum was isolated by centrifugation for 10 min at 4 °C.
The results of antibody characterization were obtained from
sera of terminal bleeds.
ELISA. The method was similar to that previously de-
scribed by Wengatz et al. (1998). Microplates were coated
overnight at 4 °C with 100 µL/well of the appropriate coating
antigen concentration in 0.1 M carbonate-bicarbonate buffer
(pH 9.6). After the coated plates were washed with PBST (PBS
plus 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), 200 µL of
blocking solution (0.5% BSA in PBS) was added and incubated
for 30 min at room temperature. After another washing step,
50 µL/well antiserum diluted in PBS with 0.2% BSA and 50
µL/well of inhibitor solution were added. The plate was
incubated for 1 h and then washed for 8-10 times. GAR-HRP
(diluted 1:3000 in PBST, 100 µL/well) was added and incubated
for 1 h at room temperature. Following another washing step
(8-10 times), tetramethylbenzidine (TMB) substrate solution
(100 µL/well; 3.3 µL of 30% H2O2, 400 µL of 0.6% TMB in
DMSO per 25 mL of acetate buffer, pH 5.5) was added. The
color development was stopped after 15-20 min with 2 M
H2SO4 (50 µL/well), and absorbances were measured at 450-
650 nm. All experiments were conducted in triplicate or
quadruplicate. Standard curves were obtained by plotting
absorbance against the logarithm of analyte concentration. The
curves were fitted to a four-parameter logistic equation: y )
{(A - D)/[1 + (x/C)B]} + D, where A is the maximum
absorbance at no analyte present, 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.
Solid -P h a se Extr a ction (SP E). An SPE method was
developed for isolating esfenvalerate from water samples.
Esfenvalerate was extracted from water using a C18 SPE
column (10 cm3/500 mg; part 1211-3027, Varian Sample
Preparation Products, Harbor City, CA). The column was
preconditioned with methanol (3.5 mL) and deionized water
(3.5 mL) 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 the water had passed through, the column was
dried under vacuum for 15 min and then eluted with 100%
methanol (3.5 mL). After one drop of propylglycol was added,
this methanol extract was evaporated to dryness under a
gentle stream of nitrogen and the residue was redissolved in
1 mL of methanol. An aliquot was analyzed by ELISA diluted
with 50% methanol in PBS (1:4).
Recover y of SP E. Radiolabeled fenvalerate (14C) was used
for the investigation of esfenvalerate SPE recovery and elution
behavior in both C8 and C18 columns. Extraction procedures
similar to those described above were used. Each blank water
sample (200 mL) was spiked with 1.35 nmol of 14C-fenvalerate
(45000 dpm). After preconditioning, extraction, washing, and
column drying steps, fenvalerate was eluted with 100%
methanol (five fractions × 3.5 mL). Each fraction was collected
in a different tube. To monitor fenvalerate losses after extrac-
tion, the filtrate (postcolumn water solution) was collected and
extracted with ethyl acetate (3 × 10 mL). This combined ethyl
acetate extract was assigned as solution A. The residue
fenvalerate on the surface of glass flask (container) was
extracted by rinsing the container with ethyl acetate, which
was assigned as solution B. Each of final eluate fractions and
surface washing and postcolumn extraction solutions were
evaporated to dryness (one drop of propylglycol was added to
each tube before evaporation) under nitrogen, and then
residues were dissolved into Scintiverse BD cocktail (3 mL)
prior to analysis by a 1409 liquid scintillation counter (Wallac,
Gaithersburg, MD).
RESULTS AND DISCUSSION
Ha p ten Syn th esis. Fenvalerate insecticide contains
four chiral isomers, a result of its two chiral centers.
The commercially produced esfenvalerate (such as
Asana) is primarily the active S,S isomer, where the
first and second assignments represent the absolute
chirality of the acid and cyanohydrin centers, respec-
tively. For this reason, and to facilitate synthesis and
purification, the (S)-4-chloro-R-(1-methylethyl)benzene-
acetic acid was used for the synthesis of the haptens.
This acid was resolved using the R and S isomers of
R-methylbenzylamine according to a modified literature
procedure (Friend, 1985). The optical purities were
determined by conversion to the amide of (S)-R-meth-
ylbenzylamine having an assigned purity of 99.6% ee
(Aldrich). Analysis by GLC gave baseline separation of
enantiomer pairs. Conversion via the acid chloride and
via DCC coupling gave nearly identical results and
indicated optical purities for the S and R isomers to be
a minimum of 96 and 98% ee, respectively.
The synthesis routes for the haptens are summarized
in Scheme 1. Haptens 4 and 7 were synthesized via
acylation of the cyanohydrins of the aldehydes 2 (Loewe
and Urbanietz, 1967) and 5 (Wengatz et al., 1998) to
give the intermediates 3 and 6 as mixture of the
diastereoisomeric pairs. A sample of the mixture, 3, was
separated into the S,S and S,R isomers by radial
chromatography. The absolute configurations were de-
termined via a stannous chloride reduction of the lower
Rf isomer to the aminofenvalerate, which was diazo-
Cr oss-Rea ctivity. The compounds listed in Tables 5 and
6 were tested for cross-reactivity by preparing each compound
in 50% methanol in PBS and determining the I50 in the ELISA.
Cross-reactivity values were calculated as follows:
CR% ) (I50 of esfenvalerate/I50 of tested compound) × 100
Assa y Op tim iza tion . Esfenvalerate standard curves were
prepared in PBS buffer containing 0, 10, 25, 50, 75, and 100%
(v/v) methanol or DMSO to determine the effects of solvent.
Curves also were prepared in PBS buffer containing 0 and
0.05% of Tween 20 to determine the effect of detergent.

Esfenvalerate Immunoassay
J. Agric. Food Chem., Vol. 47, No. 5, 1999 2151
Ta ble 1. Titr a tion Su m m a r y for An ti-esfen va ler a te An tiser a
coating antigen
10-OVA 11-BSA
antibody/immunogen
4-BSA
7-BSA
10-BSA
11-OVA
13-BSA
13-OVA
AS7584/4-LPH
AS7585/4-LPH
AS7586/4-LPH
AS7588/7-LPH
AS7589/7-LPH
+++a,b
++
+
++
+++b
++b
++
++
++
+++
+
+
-
-
+
+
++b
+
+
-
-
-
+++b
+++
+
+
+++
+
+
++
+
+
++
-
-
++
+
-
++
+
a
The data shown were determined at a coating antigen concentration of 1.0 µg/mL and an antibody dilution of 16000. (-) Absorbance
b
< 0.25; (+) absorbance ) 0.25-0.50; (++) absorbance ) 0.50-0.75; (+++) absorbance > 0.75. Homologous format.
Ta ble 2. Selected Com p etitive ELISA Resu lts for Scr een in g
combinationa
Ab/cAg
coating antigen
antiserum
(dilution)
(µg/mL)
Amax (A)
slope (B)
I50 (µg/L) (C)
Amin (D)
R2
7586/4-BSA
7586/10-BSA
7586/7-BSA
7588/4-BSA
7588/10-BSA
7588/7-BSA
0.5
0.5
0.5
0.5
0.5
0.5
1:5000
1:5000
1:5000
1:6000
1:8000
1:8000
0.59
0.68
0.24
0.47
0.63
0.66
0.98
0.80
0.83
0.81
0.61
0.81
285
296
74
158
102
29
0.03
0.04
0.01
0.03
0.04
0.06
0.99
1.00
1.00
0.99
1.00
1.00
a
Other combinations with I50 > 500 µg/L are not listed in this table. Analytes were prepared in 50% methanolic PBS solution.
tized, then reduced with hypophosphorous acid to give
(S,S)-fenvalerate identical to an authentic sample. A
similar stannous chloride reduction of 3 as the diaste-
reoisomeric pair gave hapten 4 also as a mixture of
isomers. This latter mixture was used for protein
coupling because some isomerization of esfenvalerate
and similar pyrethroids is known to occur under field
conditions. Thus, detection of both is desirable. Cleavage
of the benzyl ester 6 with iodotrimethylsilane gave the
hapten 7 having a terminal propanoic acid for protein
coupling. The deltamethrin hapten corresponding to
hapten 7 was recently reported (Queffelec et al., 1998)
synthesized according to a slightly different route.
Haptens 10 and 11 lack the terminal phenyl group in
fenvalerate and contain, respectively, acetate and hex-
anoate chains linked to the m-phenoxy group. Hapten
10 was conjugated as the S,R/ S diastereoisomeric pair,
whereas hapten 11 was conjugated as the pure S,S
isomer. Compound 13 was prepared as a hapten for the
glycine conjugate of fenvalerate acid, which is proposed
as a major metabolite of fenvalerate in humans (Miya-
moto et al., 1981). This was prepared by reaction of the
acid chlorides of (S)-4-chloro-R-(1-methylethyl)benzene-
acetic acid with 4-nitro-L-phenylalanine through a
modified Schotten-Baumann reaction to give the N-
acylated nitro intermediate 12. Subsequent catalytic
reduction of the nitro group to the amine over palladium
catalyst gave the corresponding amino hapten 13. This
reduction resulted in some dechlorination; however, the
product was easily purified by flash chromatography
and crystallization from ether to give 13 as a 1:l
etherate.
Because the phenoxybenzyl (PB) group is common to
many of the synthetic pyrethroids, the strategy for
choosing the immunogen hapten was to link the protein
through the aromatic end of the esfenvalerate molecule
to minimize the contribution of PB to antibody specific-
ity. Therefore, LPH conjugates of haptens 4 and 7 were
used for antibody production, and their BSA conjugates
and conjugates of other haptens served as coating
antigens.
The hapten density (the number of hapten molecules
per molecule of protein) of conjugates was estimated
indirectly by competitive ELISA (Wengatz et al., 1998).
The hapten densities for 4-BSA and 7-BSA are 28 and
22, respectively. Because the antibodies used in this
study were raised against LPH conjugates, their hapten
densities could not be measured by using this method.
The LPH protein molecule is ∼5 times bigger than BSA,
and it might have more free amine or carboxylic acid
residues on the protein surface. By using the same
coupling methodology, the immunogens 4-LPH and
7-LPH would have a higher hapten density than their
BSA conjugates.
Scr een in g of An t iser a . Titers of all five antisera
were tested against eight different coating antigens
using a checkerboard titration system (Gee et al., 1994).
The results of the titration experiments with the
terminal bleeds are shown in Table 1. All raw antisera
showed higher titers in a homologous system, in which
the same hapten (but a different carrier protein) was
used for both immunogen and coating antigen (cAg),
than in heterologous systems. These results are consis-
tent with a previous study on the pyrethroid fenpro-
pathrin by Wengatz et al. (1998). The titers were also
varied among different immunogens and rabbits. Among
the five antisera tested, antibody 7588, which was
generated against antigen 7-LPH, exhibited the high-
est titer with all coating antigens. Only those combina-
tions of antiserum and coating antigen with OD absor-
bance of >0.50 in Table 1 were screened for competition
by esfenvalerate. The I50 values ranged from 30 to 300
µg/L in the homologous and heterologous systems tested
(Table 2). The homologous system for Ab7588 (with cAg
7-BSA) showed the lowest I50 (29 µg/L), which was at
least 4 times better than the heterologous format.
However, a heterologous system worked best for Ab7586
(with cAg 7-BSA, I50 ) 74 µg/L), which was ∼4 times
more sensitive than the homologous one. In this study,
only the homologous system of Ab7588 and cAg 7-BSA
was used for further assay development.
Op tim iza tion . The effects of the solvents (methanol
or DMSO) and detergent on the ELISA system (Ab7588/
7-BSA) were evaluated by preparing esfenvalerate in
a buffer containing various amounts of solvent or Tween
20. The results (Tables 3 and 4) showed that these
solvents significantly influence assay sensitivity and
absorbance. The maximum absorbance (without ana-
lyte) was enhanced with increasing MeOH concentra-
tion. However, it was significantly decreased in the
presence of DMSO (Table 4), indicating that high
concentration of DMSO suppresses antibody-hapten

2152 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Shan et al.
Ta ble 3. Effects of Meth a n ol Con cen tr a tion a
MeOHb
(%)
slope
(B)
I50 (µg/L)
(C)
Amax (A)
A
min (D) A/D R2
0
10
25
50
75
0.61 ( 0.02c 0.89 247 ( 24.2c
0.09
0.10
0.08
0.09
0.18
0.31
6.3 0.99
6.2 1.00
8.8 0.99
9.3 1.00
5.0 1.00
3.5 0.99
0.65 ( 0.03
0.71 ( 0.03
0.84 ( 0.02
0.90 ( 0.04
0.95 ( 0.04
0.78 155 ( 9.2
0.74
0.83
1.08
68 ( 10.0
32 ( 5.5
41 ( 6.6
100
1.07 117 ( 17.0
a
ELISA conditions: coating antigen 7-BSA (0.4 µg/mL); an-
b
tiserum 7588 (1:6000); goat anti-rabbit IgG-HRP (1:3000). Con-
centration of methanol in esfenvalerate standard solution (PBS/
MeOH). c Mean value ( SD. Each set of data represents the
average of three experiments.
Ta ble 4. Effects of DMSO Con cen tr a tion a
DMSOb
(%)
slope
(B)
I50 (ppb)
(C)
Amax (A)
A
min (D) A/D R2
F igu r e 4. ELISA inhibition curve for esfenvalerate, using
antiserum 7588 (diluted 1:12000, final dilution in well), and
coating antigen 7-BSA (0.4 µg/L). This standard curve rep-
resents the average of 25 curves.
0
10
25
0.61 ( 0.02c 0.89 247 ( 24.2c
0.09
0.06
0.06
6.3 0.99
7.0 1.00
6.2 0.99
0.45 ( 0.04
0.37 ( 0.03
0.70 207 ( 32.0
0.76 219 ( 18.5
a
ELISA conditions: coating antigen 7-BSA (0.4 µg/mL); an-
Ta ble 5. Cr oss-Rea ctivities of F en va ler a te Isom er s a n d
Oth er Str u ctu r a lly Rela ted Com p ou n d s
b
tiserum 7588 (1:6000); goat anti-rabbit IgG-HRP (1:3000). Con-
centration of DMSO in esfenvalerate standard solution (PBS/
DMSO). c Mean value ( SD. Each set of data represents the
average of three experiments.
% cross-
analyte
R
reactivitya (n)
fenvalerate isomers
1S,2S (esfenvalerate)
1S,2R
1R,2S
1R,2R
1S,2R/S
1R,2R/S
1R/S,2R/S (fenvalerate) Ph
haptens
Ph
Ph
Ph
Ph
Ph
Ph
100 (9)b
93 ( 7 (3)
14 ( 4 (3)
1.8 ( 0.8 (3)
97 ( 3 (3)
14 ( 2 (3)
60 ( 9 (3)
4
6
7
8
10
9
11
4-PhNH2
750 ( 85 (3)
4-PhCH2CH2COOCH2Ph 44 ( 9 (3)
4-PhCH2CH2COOH
CH2COOCH2Ph
CH2COOH
CH2(CH2)4COOCH2Ph
CH2(CH2)4COOH
2050 ( 220 (3)
143 ( 35 (3)
610 ( 110 (3)
39 ( 20 (3)
4000 ( 330 (3)
F igu r e 3. Effect of detergent (Tween 20): (O) 0%; (b) 0.05%.
a
Cross-reactivity was calculated as (I50 of esfenvalerate/I50 of
b
analyte) × 100%. n, number of times cross-reactivity experiment
binding. Consequently, only MeOH was chosen for
further optimization. The optimal MeOH concentration
was selected on the basis of I50 values and the ratios of
maximum and minimum absorbances for esfenvalerate
standard curves (A/D). Because esfenvalerate is highly
lipophilic and will adhere to glass and plastic surfaces
(Sharom and Solomon, 1981), higher concentrations of
MeOH might reduce such losses by solution of adsorbed
material. The lowest I50 values were measured at 50 and
75% MeOH (32 and 41 µg/L, respectively). Because of a
low A/D ratio for 75% MeOH, a MeOH concentration of
50% was selected for subsequent experiments.
Tween 20, a nonionic detergent, is commonly used in
many immunoassays to reduce nonspecific binding and
improve sensitivity (Vanderlaan et al., 1988; Chiu et al.,
1995). In this study, Tween 20 significantly affected the
binding between antibody and hapten (Figure 3). At the
concentration tested (0.05% of Tween 20), the I50 with
Tween 20 was ∼20 times higher than that without the
detergent. The maximum absorbance was significantly
suppressed in addition. This is probably caused by
nonspecific hydrophobic interaction between detergent
and lipophilic esfenvalerate molecules in an aqueous
was conducted.
system (Manclus and Montoya, 1996; Sugawara et al.,
1998). Therefore, no Tween 20 was used in either the
analyte solution or the antiserum solution in this assay.
The optimized esfenvalerate ELISA used 0.4 µg/mL
of coating antigen 7-BSA, antibody 7588 at a dilution
of 1:6000, and esfenvalerate in MeOH/PBS buffer (1:1).
The I50 value of this homologous assay was 30 ( 6.2
µg/L (Figure 4) with a lower detection limit (LDL) of
3.0 ( 1.8 µg/L. The LDL was estimated as the concen-
tration that corresponded to the absorbance of the
control (zero concentration of analyte) minus 3 times
the standard deviation of the control (Grotjan and Keel,
1996).
Cr oss-Rea ctivity. The antibody from rabbit 7588
was highly selective for esfenvalerate, and its ability to
bind with fenvalerate depended upon the isomer ana-
lyzed (Table 5). In an optimized homologous system
(Ab7588/7-BSA), no significant cross-reactivity was
measured for most compounds tested, including pyre-
throids and fenvalerate metabolites (Table 6). It is of
importance that several widely used, PB-containing

Esfenvalerate Immunoassay
J. Agric. Food Chem., Vol. 47, No. 5, 1999 2153
Ta ble 6. Cr oss-Rea ctivities of P yr eth r oid s a n d
Esfen va ler a te Meta bolites
Ta ble 7. SP E Meth od Develop m en t
14C-fenvalerate recovereda (SD)
C8 column (n ) 3) C18 column (n ) 3)
%
% cross-
% cross-
analyte
reactivitya
analyte
reactivitya
eluatea
fraction 1
fraction 2
fraction 3
fraction 4
fraction 5
82 ( 3.1
0.48 ( 0.16
0.25 ( 0.08
0.11 ( 0.04
<0.05
85 ( 3.9
esfenvalerate
100
<0.01
<0.01
nib
phenothrin
cyfluthrin
fenpropathrin
resmethrin
fluvalinate
ni
ni
ni
ni
ni
1.05 ( 0.15
0.31 ( 0.08
0.17 ( 0.04
0.11 ( 0.01
13 ( 2.7
phenoxybenzoic acid
(S)-fenvalerate acid
permethrin
cypermethrin
ni
solution Ab
solution Bc
13 ( 2.2
deltamethrin
ni
2.2 ( 0.20
<0.05
a
Cross-reactivity was calculated as (I50 of esfenvalerate/I50 of
a
Analyte was eluted with 100% methanol, 3.5 mL each fraction.
b
analyte) × 100%. ni, <10% inhibition was measured at highest
b
c
Solution washed from glass container. Solution extracted from
concentration (10000 µg/L).
postcolumn solution (filtrate).
pyrethroids such as permethrin, cypermethrin, and
deltamethrin do not interfere with this assay, which
makes this technique very useful for the selective
detection of this compound. Compared to (S,S)-fenval-
erate as a reference, Ab7588 fully recognized the S,R
enantiomer of fenvalerate, and the S,R/S pair of dias-
tereoisomers, which have the same absolute configura-
tion as immunogen hapten. However, isomers that have
the R configuration at the acid portion of the fenvalerate
molecule have a very low cross-reactivity (<14%). This
is consistent with the study by Wing and Hammock
(1979) in which their immunoassay showed the highest
selectivity for the optical center most distal to the site
of coupling. The data indicate that in this study the
stereostructure of the acid moiety of fenvalerate is more
critical than that of alcohol portion in determining
antibody stereoselectivity. These further supported the
hypothesis that the portion of hapten farthest from the
protein is the most important in causing antibody
specificity (Parker, 1976; Wing and Hammock, 1979).
It must be pointed out that the (S)-fenvalerate acid used
in the synthesis of the haptens contained ∼1% of R
isomer (see synthesis above). If this carried through the
synthetic steps to the final haptens, it would generate
some antibodies to the R,R/S isomers and thus contrib-
ute to the cross-reactivity.
Finally, cross-reactivity experiments were conducted
for haptens, which were used for preparing antigens
(Table 5). A high inhibition, ∼25 times better than with
esfenvalerate, was detected for immunogen hapten 7.
Similar phenomena have been reported (Wing et al.,
1978; Stanker et al., 1987) for (S)-bioallethrin and dioxin
immunoassays, respectively, in which hapten handle
recognition was assumed to be a contributing factor.
However, this assay was also extremely sensitive to
haptens 4, 10, and 11, which have handles different
from that of the immunogen hapten. Esfenvalerate is a
very lipophilic compound, which may limit the interac-
tion between antibody and analyte. In addition, haptens
4, 7, 10, and 11 all contain -NH2 or -COOH groups,
which make these compounds more polar and more
soluble in aqueous system. Therefore, the solubility of
those haptens could be a contributing factor for their
high cross-reactivity (Stanker et al., 1987). This hypoth-
esis is also supported by the fact that the cross-reactivity
was significantly lowered when those -COOH-contain-
ing haptens (4, 10, and 11) were esterified as benzyl
esters (Table 5).
Richard, 1988; Swineford and Belisle, 1989; Durhan et
al., 1990). A recent study by Woin (1994) showed that
C8 sorbent is as effective as C18 for pyrethroid extraction,
showing very good recoveries (80-100%). In the present
study, high recovery rates were achieved for both C8 and
C18 columns (82 and 85%, respectively) when applied
to tap and river water samples (Table 7). Although the
C8 column had higher eluting efficiency with methanol
than the C18 column (0.84 versus 1.6% of analyte in
second through fifth eluate fractions), the C18 column
is much more effective for extracting fenvalerate from
water than the C8 column (<0.05 versus 2.2% of analyte
lost in filtrate). To ensure a higher rate of recovery, a
C18 SPE column was used in this assay. Because
fenvalerate can be easily adsorbed to glass and plastic
materials (Schoor and McKenney, 1983; Day, 1991;
Woin, 1994), sample handling is of great importance for
the accurate detection of this compound in water. In this
study, only 13 ( 2.7% of fenvalerate was found to adhere
to the surface of the glass container, which is fairly low
compared with previous studies (Schoor and McKenney,
1983; Day, 1991). In our experience, to maintain a
consistently low level of adsorption, a thorough rinse of
the glass containers with deionized water is required.
This reduces adsorption by 6-8% (data not shown).
Adding a trap solvent such as propylglycol is also very
helpful. Finally, sonication was proposed when the
water samples were kept in a container for >12 h
(reducing losses by 5-10%, data not shown).
Assay validation was conducted in a blind fashion by
SPE plus ELISA for tap and Sacramento River water
samples, which were spiked with esfenvalerate concen-
trations ranging from 0 to 50 ppb (Figure 5). Good
correlation between spiked and ELISA-measured es-
fenvalerate was obtained from linear regression analysis
(Y ) 0.98x - 0.079, R2 ) 0.96, n ) 16). All recoveries
were >80% of spiked value. Recovery of esfenvalerate
for five water samples spiked at 0.1 µg/L was 0.102 (
0.018 µg/L (102 ( 18%). These results demonstrate that
this ELISA is suitable for the detection of esfenvalerate
in water contamination monitoring, which requires high
sensitivity.
In conclusion, careful hapten design and preparation,
extensive studies with various antibody-antigen com-
binations, and optimization of an ELISA resulted in a
homologous immunoassay (Ab7588/7-BSA) that is highly
selective and fairly sensitive for esfenvalerate. With a
rapid and simple SPE step, this ELISA was successfully
applied to the quantitative detection of sub parts per
billion amounts of esfenvalerate in water. A high
concentration of cosolvent (methanol) in this ELISA
system (50% in analyte and standard solution) is
extremely important for the accurate performance of the
ELISA for the highly lipophilic esfenvalerate. Finally,
SP E a n d Assa y Va lid a tion . To detect esfenvalerate
in water samples at a sub parts per billion level, a rapid
and efficient SPE procedure was developed. The C18
column has been considered the primary choice for
extracting nonpolar or semipolar pesticides including
pyrethroids from natural water sources (J unk and

2154 J. Agric. Food Chem., Vol. 47, No. 5, 1999
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ABBREVIATIONS USED
Ab, antibody; BSA, bovine serum albumin; BSTFA,
bis(trimethylsilyl)trifluoroacetamide; cAg, coating an-
tigen; DAPEC, 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide; DCC, 1,3-dicyclohexylcarbodiimide; DMF,
N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
ELISA, enzyme-linked immunosorbent assay; FAB-
HRMS, fast atom bombardment high-resolution mass
spectrum; GAR-HRP, goat anti-rabbit immunoglobulin
conjugated to horseradish peroxidase; GLC, gas-liquid
chromatography; HPLC, high-performance liquid chro-
matography; I50, the concentration of analyte giving 50%
inhibition; LDL, lower detection limit; LPH, hemocyanin
from Limulus polyphemus; Morpho-CDI, 1-cyclohexyl-
3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene-
sulfonate; NHS, N-hydroxysuccinimide; NMR, nuclear
magnetic resonance; OVA, ovalbumin; PB, phenoxy-
benzyl; PBS, phosphate-buffered saline; PBST, phos-
phate-buffered saline with 0.05% of Tween 20; RT, room
temperature; SPE, solid-phase extraction; THF, tet-
rahydrofuran; TLC, thin-layer chromatography; TMB,
tetramethylbenzidine.
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454.
ACKNOWLEDGMENT
We thank A. Daniel J ones (Pennsylvania State Uni-
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for mass spectral determinations and the Department
of Chemistry (UCD) for the use of their NMR instru-
ment.
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received March 16, 1999. Accepted March 19, 1999. This
project was funded by the National Institute of Environmental
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