Metabolism of (S)-bioallethrin and related compounds in humans

Article abstract of DOI:10.1016/S0378-4274(99)00038-7

Chrysanthemate insecticides like (S)-bioallethrin, natural pyrethins, and related pyrethroids are subjected to extensive hydrolytic and oxidative degeneration by the mammalian metabolism, leading to a complex series of metabolites partially conjugated and finally eliminated in the urine. The major oxidation products of chrysanthemic acid, cis-(E)- and trans-(E)-chrysanthemumdicarboxcylic acid (cis-(E) and trans-(E)-CDCA), were synthesized and their structures were established by nuclear magnetic resonance spectrometry (H¹-NMR) and mass spectrometry (MS). Diastereoselective separation was by high performance liquid chromatography (HPLC) and capillary gas chromatography (GC). An analytical method for extraction and identification of CDCA from human urine was developed. Quantitation was by gas chromatography and electron-impact mass spectrometry (GC/MS). The limit of detection was 20 μg/l for cis-(E)-CDCA and 10 μg/l for trans-(E)-CDCA. To test the applicability of the presented method, urine samples of humans exposed to (S)-bioallethrin were investigated. Urinary peak excretion of trans-(E)-CDCA occurred within 24 h after exposure. Copyright (C) 1999 Elsevier Science Ireland Ltd.

Full text of DOI:10.1016/S0378-4274(99)00038-7

Toxicology Letters 107 (1999) 109–121
Metabolism of (S)-bioallethrin and related compounds in
humans
Gabriele Leng *, Karl-Heinz K u¨ hn, Brigitte Wieseler, Helga Idel
Institute of Hygiene, Heinrich–Heine Uni6ersity D u¨ sseldorf, Moorenstraße 5, 40225 D u¨ sseldorf, Germany
Accepted 31 January 1999
Abstract
Chrysanthemate insecticides like (S)-bioallethrin, natural pyrethins, and related pyrethroids are subjected to
extensive hydrolytic and oxidative degeneration by the mammalian metabolism, leading to a complex series of
metabolites partially conjugated and finally eliminated in the urine. The major oxidation products of chrysanthemic
acid, cis-(E)- and trans-(E)-chrysanthemumdicarboxcylic acid (cis-(E) and trans-(E)-CDCA), were synthesized and
1
their structures were established by nuclear magnetic resonance spectrometry (H -NMR) and mass spectrometry
(
MS). Diastereoselective separation was by high performance liquid chromatography (HPLC) and capillary gas
chromatography (GC). An analytical method for extraction and identification of CDCA from human urine was
developed. Quantitation was by gas chromatography and electron-impact mass spectrometry (GC/MS). The limit of
detection was 20 mg/l for cis-(E)-CDCA and 10 mg/l for trans-(E)-CDCA. To test the applicability of the presented
method, urine samples of humans exposed to (S)-bioallethrin were investigated. Urinary peak excretion of trans-(E)-
CDCA occurred within 24 h after exposure. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: (S)-Bioallethrin; Chrysanthemumdicarboxylic acid; Human metabolism; Synthesis; Biomonitoring method
Nowadays, pyrethrum is formulated as aerosol or
spray with synergists (e.g. piperonyl butoxide) for
the use as a rapid knock-down or flushing agent
in industry and in the home.
Purified pyrethrins are non-polar, viscous liq-
uids susceptible to decomposition by ultraviolet
light, oxygen, acid, and alkali. The replacement of
specific structural elements found in pyrethrin I
with moieties designed to increase the stability in
the environment was an important strategy for
the development of the first synthetic pyrethroids
like allethrin. In such early analogs, the chrysan-
1
. Introduction
The synthetic pyrethroid insecticides have their
origin in the important botanical insecticide
pyrethrum, an extract obtained from the flowers
of Chrysanthemum cinerariaefolium. The six natu-
ral esters of pyrethrum (pyrethrins, jasmolins and
cinerins) are effective against a wide range of
household and public health insects (Fig. 1).
*
Corresponding author. Tel.: +49-211-8112607; fax: +49-
2
11-8112919.
E-mail address: leng@uni-duesseldorf.de (G. Leng)
themic acid moiety (CA; X=CH ) was kept in-
3
0
378-4274/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0378-4274(99)00038-7

1
10
G. Leng et al. / Toxicology Letters 107 (1999) 109–121
tact and only a minor change in the molecular
structures of esterifying alcohols were made.
Thus, in contrast to the natural pyrethrins, al-
lethrin is less volatile and more stable to heat and
light.
thermore, (S)-bioallethrin has served as an impor-
tant model compound in numerous animal studies
(Elliott et al., 1972; Class et al., 1990; Ando et al.,
1991a).
Based on the binding assay on the animal
gamma-aminobutyric acid (GABA) receptor-
ionophore complex, ester pyrethroids are divided
in two major classes: Type I pyrethroids without
an a-cyano group and Type II pyrethroids bear-
ing an a-cyano-3-phenoxybenzyl group in the al-
cohol moiety (Gammon et al., 1982; Lawrence
and Casida, 1983). Like the parent compound
allethrin, (S)-bioallethrin is classified as Type I
pyrethroid. The Type I or ‘T’-syndrome involves
hyperactivity, tremor, convulsion and paralysis in
mammals and insects. The primary mechanism of
neurotoxic action is determined by an extended
opening of the sodium channel in the nerve mem-
branes, leading to prolonged depolarization and
induction of repetitive nerve impulses (Aldrige,
1990; Vijverberg and van den Bercken, 1990; Na-
harashi, 1992). The overall results of such a tran-
sient increase in the sodium permeability is
associated with pronounced repetitive activity in
sense organs and in sensory nerve fibers (Van den
Bercken, 1977). While all pyrethroids have essen-
tially the same basic mechanism of action, how-
ever, the rate of relaxation differs substantially for
The presence of two chiral centers (C-1 and
C-3) in the cyclopropane ring of CA produces two
pairs of diastereomers, commonly designed cis or
trans based on the orientation of the substituents
in relation to the plane of the cyclopropane ring
(
Figs. 1 and 2). Whereas the acid moieties of the
natural pyrethrins are exclusively in the [1R,
trans] configuration, esters of synthetic CA were
initially evaluated as racemic mixtures. Beside the
stereochemistry of the acid moiety, the chiral cen-
ter (C-1%) in the alcohol group plays also a signifi-
cant role for
a high insectical activity of
pyrethroids. Among the possible eight stereoiso-
mers in the racemic mixture of allethrin, (S)-
bioallethrin ([1R, trans; 1%S]) is the most
biologically active followed by the [1R, cis; 1%S]-
isomer (WHO, 1989a). Consequently, (S)-bioal-
lethrin is several times more effective against
crawling and flying insects as allethrin or the
natural pyrethrins.
This substantial increase in potency is responsi-
ble for the use of a single stereoisomer as active
ingredient in many insecticide formulations. Fur-
Fig. 1. Structures of pyrethrin I, pyrethrin II (only trans configuration of CA and PA are found in nature) and allethrin (racemic
mixture of cis-/trans-CA).

G. Leng et al. / Toxicology Letters 107 (1999) 109–121
111
Fig. 2. Biooxidation of the isobutenyl moiety in the tentative metabolic pathway for (S)-bioallethrin in humans.
the various compounds. Allethrin and related
pyrethroids are known to cause a transient itching
or burning sensation in human skin (paresthesia).
However, due to the poor oral and dermal
uptake and followed by a rapid metabolism, these
insecticides are relatively safe for birds and mam-
mals. The acute oral toxicity of allethrin and its
isomers for rats is moderate to weak (LD : 378–
sive use indoors, especially in narrow and poorly
ventilated spaces has resulted in elevated insecti-
cide concentrations, bearing a potential source of
human exposure (Class and Kintrup, 1991).
Generally, when chrysanthemate insecticides
are administered to mammals, detoxification takes
place in part by hydrolysis, oxidation and finally
conjugation, transforming the compounds into
more water soluble urinary metabolites (Elliott et
al., 1972; Class et al., 1990).
5
0
2430 mg/kg body weight; WHO, 1989b).
Nevertheless, human exposure will be mainly
through inhalation, often in combination with an
oral uptake of residues of aerosol sprays and from
other household applications (e.g. electro mate,
mosquito coil). It is anticipated that their exten-
In previous animal studies, ester hydrolysis and
oxidation of the (E)-methyl group of the
isobutenyl side chain have been identified as ma-
jor metabolic pathways of CA (Elliott et al., 1972;

1
12
G. Leng et al. / Toxicology Letters 107 (1999) 109–121
Ueda et al., 1975; Brown et al., 1985) leading to
the formation of the corresponding chrysanthe-
mumdicarboxylic acid (CDCA) in high yields
2.2. Chemicals
The ethyl esters of cis/trans-CA, 2-phenoxyben-
zoic acid (2-PBA), selen dioxide, activated man-
ganese dioxide, sodium cyanid, 3,3-dimethyl-
acrolein (b-methyl-crotonaldehyde), ethyl a-bro-
mopropionate, ethyl diazoacetate, tert. butyl
methyl ether, ligronin, zinc, and copper powder
were purchased from Sigma–Aldrich (Steinheim,
Germany). (S)-bioallethrin and the natural
pyrethrins were from Ehrensdorfer (Augsburg,
Germany), potassium hydroxide, hexane, toluene,
ethanol, methanol and propanol were from Merck
(
Fig. 2). Furthermore, trans-(E)-CDCA is also a
major product of the hydrolysis of pyrethric acid
PA; X=CO CH ) and related natural esters of
(
2
3
pyrethrum (pyrethrin II, jasmolin II and cinerin
II) (Straudinger and Ruzicka, 1924; Crombie et
al., 1957, 1970).
This is in contrast to the metabolism of suffi-
cient photostable pyrethroids (e.g. permethrin,
cyfluthrin, cypermethrin or deltamethrin) whereas
the isobutenyl moiety is replaced by a halovinyl
group. After a human exposure to these insecti-
cides a significant amount of the unchanged halo-
genated acid moiety is present in the urine
(
Darmstadt, Germany), conc. hydrochloric and
conc. sulphuric acid were purchased from Baker
Deventer, The Netherlands), all other chemicals
were from Promochem (Wesel, Germany).
(
(
Eadsforth et al., 1988; Woollen et al., 1992;
K u¨ hn et al., 1996; Leng et al., 1997a,b).
Moreover, oxidation and epoxidation followed
by hydration of the alcohol moiety of (S)-bioal-
lethrin leads to a series of more complex metabo-
lites. Nevertheless, these epoxy and hydroxy
derivatives are clearly unsuitable for a human
biomonitoring without further study as they ex-
hibited a remarkable species specificity in their
metabolism (Class et al., 1990; Ando et al.,
2
.3. Synthesis of cis-(E)- and
trans-(E)-chrysanthemumdicarboxcylic acid
The required metabolite trans-(E)-CDCA and
its cis-(E) isomer were prepared by two indepen-
dent methods:
(
A) A mixture of ethyl cis/trans-chrysanthemate
2.0 g of cis/trans-CA-Et) with selen dioxide (1.6
g of active SeO ) in ethanol (20 ml) refluxed for
(
1991b).
2
Such precedents suggests that the acid moiety in
1
h lead to the formation of the corresponding
chrysanthemate insecticides is metabolized in hu-
mans and mammals by similar hydrolytic and
oxidative routes, leading to the formation of
CDCA as an important biomarker of internal
burden.
aldehyde (1.0 g of cis/trans-[(E)-CHO]-CA-Et,
Figs. 3 and 4) possessing only E configuration in
the isobutenyl group (Crombie et al., 1970; Brown
et al., 1985). The obtained aldehyde (0.1 g of
cis/trans-[(E)-CHO]-CA-Et) was stirred with a
mixture of sodium cyanid (0.08 g), acetic acid
2. Materials and methods
(
0.03 g), and manganese dioxide (0.6 g) in 10 ml
ethanol for 16 h at room temperature (Corey et
al., 1968). After hydrolysis of the diethyl chrysan-
themum-dicarboxylates (Crombie et al., 1957), the
formatted diacids were subjected to purification
and diastereoselective separation by preparative
2
.1. Designations
The structures of (S)-bioallethrin, trans-
chrysanthemic acid (trans-CA) and its oxidation
product trans-(E)-chrysanthemumdicarboxcylic
high
performance
liquid
chromatography
acid (trans-(E)-CDCA) are given in Fig. 2. The
stereoisomers about the cyclopropane ring are
designated as cis or trans and of the side chain
from oxidation of the chrysanthemate isobutenyl
methyl groups as E. In the older literature the E
stereochemistry of the olefinic linkage is often also
(HPLC).
The overall yield of (E)-trans-CDCA and (E)-
cis-DCCA was in the order of 42%.
(B) An alternative route to avoid the toxic selen
dioxide starts with a Reformatski reaction be-
tween b-methylcrotonaldehyde (11.0 g), ethyl a-
indicated as trans (Crombie et al., 1957).
o

G. Leng et al. / Toxicology Letters 107 (1999) 109–121
113
bromopropionate (25.0 g) and zinc (9.0 g) in
toluene (25 ml) yielding the conjugated ethyl 2,5-
in the same order of magnitude and have been
proofed to be spectroscopically (gas chroma-
tography and mass spectrometry (GC/MS)) iden-
tical with the separated diacids of the first prepa-
ration. The latter method has the additional
benefit to prepare larger quantities of DCCA
more easily.
The hydrolysis of natural pyrethrins with potas-
sium hydroxide (5 g) in ethanol (50 ml) gave a
mixture of trans-CA and trans-(E)-CDCA (Crom-
bie et al., 1970). Subsequent esterification of the
mixture with ethanol produced the corresponding
esters having identical chromatographical proper-
dimethylhexa-2,5-dienoate
(2,5-dimetylsorbate)
(
8.7 g) (Crombie et al., 1957). In a second step,
2,5-dimetylsorbate (8.5 g) and ethyl diazoacetate
(
10.7 g) in ligroin (20 ml) were used in an copper-
catalyzed condensation (0.5 g of Cu) to build up
the desired cyclopropane derivatives of CDCA
(
6.0 g of cis-(E)/trans-(E)-DCCA) (Harper and
Reed, 1955; Crombie et al., 1957; Ettlinger et al.,
957). Again, after hydrolysis, diastereoselctive
1
separation, and purification using preparative
HPLC, the overall yields of the free diacids were
Fig. 3. Main fragmentations of the aldehyde (cis-/trans-[(E)-CHO]-CA-Et) formated as an intermediate from the SeO oxidation of
2
the isobutenyl methyl group of cis-/trans-CA-Et.

1
14
G. Leng et al. / Toxicology Letters 107 (1999) 109–121
Fig. 4. ITD-mass spectra (full scan 107–220 m/e) showing the main fragmentations of the aldehyde (cis-/trans-[(E)-CHO]-CA-Et)
indicated in Fig. 3.
ties and mass spectra (GC/MS) as the trans-(E)
isomer of the first and second preparation.
2.5. Diastereoselecti6e HPLC analysis
For the development of an analytical method
for the detection and quantitation of CDCA in
biological matrices methyl, ethyl and n-propyl
esters were prepared and subjected to GC/MS.
Standards were solved in the corresponding alco-
hol and esterification was accomplished by adding
concentrated sulphuric acid (K u¨ hn et al., 1996).
Using electron impact (EI) mass spectroscopy,
main fragmentations of the diesters of trans-(E)-
CDCA were compared with those of pyrethric
acid and related compounds showing similar
pathways of decay (Pattenden et al., 1972).
Solutions of the diacids were separately pre-
pared in acetonitril and an aliquot of 2.5 ml were
injected onto a HPLC (Merck–Hitachi, Ger-
many) equipped with a 3.5-mm Zorbax SB, reverse
phase C-18 steel column (Dupont De Nemours &
Co., USA, 75 mm×4.6 mm) maintained at 45°C.
The mobile phase was 0.1% phosphoric acid (A)/
acetonitril (B) with a gradient (% B): 10 (0.8 min),
from 10 to 95 in 6.7 min, 95 (1.6 min). The flow
rate was 3.0 ml/min and the column eluent was
monitored at 210 nm with a DAD-UV detector
(Gynkotek, Germany). Time of retention was 2.3
min for trans-(E)-CDCA and 2.7 min for cis-(E)-
CDCA. The purity of the separated isomers was
2.4. Preparati6e HPLC
9
1% for cis-(E)-CDCA and greater than 99% for
trans-(E)-CDCA.
For diasteroselective separation, 100 mg of a
mixture of the CDCA isomers was solved in 8 ml
water/acetonitril (70/30%) and was injected onto a
autoprep HPLC (Gilson–Abimed, USA) fitted
with a 5-mm Kromasil 100-5, reverse phase C-18
steel column (Eka Nobel, Sweden; 250 mm×20
mm) maintained at 25°C. The mobile phase was
water/acetonitril (70/30%). The flow rate was 25
ml/min and the column eluent was monitored at
1
2.6. Identification of the diacids by H NMR
The configuration of the separated isomers were
determined spectroscopically by H NMR. Spec-
1
tra were recorded on an AMX-400 (Bruker) in-
strument, using dilute solutions of CDCA in
DMSO with tetramethylsilane (TMS) as internal
standard. The cyclopropane ring protons (Fig. 2:
H-1, H-3) and the olefinic proton (Fig. 2: H-7) in
the isobutenyl moiety in both isomers constitute
ABX-type spin systems. The appearance of the
olefinic proton in the side-chain of the separated
2
10 nm. The auto separation program was set
from 5.0 to 5.4 min for the first and from 7.2 to
.3 min for the second peak fraction. The frac-
8
tions of separated isomers were concentrated un-
der reduced pressure at 45°C.

G. Leng et al. / Toxicology Letters 107 (1999) 109–121
115
diacids in the NMR spectra as a doublet of
multiples (dm) has been used for the assignment
of the overall configuration either as trans-(E)-
CDCA (d 6,44 ppm, dm) or cis-(E)-CDCA (d
holding 300°C during GC run. The split flow
was 15 ml/min. The split was open during injec-
tion, closed after 0.2 min for 2 min and re-
mained open during GC run. The column was
maintained at 70°C (1 min) and programmed
from 70°C at 20°C/min to 140°C (1 min) and
from 140°C at 10°C/min to 270°C (1 min). The
transfer-line was set at 270°C and automatic
gain control (AGC) was 29 000 ms. Electron im-
pact (EI) ionization was at 70 eV and full scan
mass chromatograms were recorded from 107 to
6,99 ppm, dm). This is in close agreement with
1
the analysis of H NMR spectra of trans-(E)-
pyrethrate and related compounds derived from
the natural pyrethrins (Bramwell et al., 1969; El-
liott et al., 1972).
2.7. Diastereoselecti6e GC/MS analysis
2
85 m/e. To enhance the sensitivity of the in-
strument multiple ion detection (MID) was from
23 to 197 m/e and from 242 to 282 m/e. Quan-
Generally, diastereoselective separation by
1
capillary gas chromatography will be achieved
for diethyl and dipropyl esters of cis-(E)- and
trans-(E)-CDCA on a non-polar DB 5 ms (sta-
tionary phase: 5% diphenyl- 95% dimethyl
polysilane) column (Table 1 and Fig. 7). After
derivatization, an aliquot (2 ml) of the prepara-
tions was injected onto a PE Autosystem GC
titation was achieved by internal calibration us-
ing 2-phenoxybenzoic acid (2-PBA) as internal
standard. 2-PBA was monitored at 197 and 242
m/e for its ethyl and at 197 and 256 m/e for its
propyl ester. Retention times and major mass
ions of the derivatives of CDCA are indicated
in Figs. 5 and 6 and Table 1.
(
Perkin–Elmer, USA) fitted with a 30-m DB 5
ms capillary column (J&W, USA; 0.25 mm i.d.,
.1 mm film) equipped with an ITD 800 ion trap
2.8. Sample collection
0
detector (Finnigan MAT, USA). The carrier gas
was helium (152 kPa). The temperature-pro-
grammable split/splittless injector Model 011
Urine samples (24 h or spot samples) of ex-
posed subjects were collected in polyethylene
bottles. Until preparation samples were stored at
(
QUMA, Germany) was set at 80°C (0.5 min)
−
21°C. Under these storage conditions, samples
and programmed from 80°C at 15°C/s to 300°C,
were stable for a minimum of 1 year.
Table 1
Key fragmentation patterns and retention times (T(R)) of the
formated derivatives of CDCA separated on a DB 5 ms
column (MID-quantitation ions with sufficient high mass-to-
charge ratios are indicated).
2.9. Analysis of CDCA in human urine samples
A volume of 2 ml of an urine sample includ-
ing the internal standard 2-PBO were placed in
a glass centrifuge tube equipped with screw cap.
After diluting with 2 ml 1 M KOH, the tightly
sealed glass was placed in a water bath (50°C)
for 1 h. The diacid metabolites and their conju-
gates were converted into free acids by adding
Derivatives
m/e
T(R) (min)
Dimethyl cis-(E)- and
trans-(E)
226, 183, 167,
135, 107
6.3
-
chrysanthemumdicar-
boxylate
Diethyl cis-(E)-chrysanthe- 209, 181, 153,
mumdicarboxylate 135, 139, 107
Diethyl trans-(E)-chrysan- 254, 181, 163,
themumdicarboxylate 153, 139, 107
Dipropyl cis-(E)-chrysan- 223, 163, 153,
0
.5 ml concentrated HCl to the urine sample
7.4
7.6
(
pHB1). The acidified urine was treated with 2
ml tert. butyl methyl ether, vigorously shaken
for 1 min and rolermixed for 5 min. Following
centrifugation (7×g, 10°C) for 10 min, the or-
ganic layer was transfered to a second centrifuge
tube. This procedure was repeated once more.
In order to reduce the urinary matrix interfer-
ence, the diacids were subjected to the following
9.4
themumdicarboxylate
Dipropyl trans-(E)-
chrysanthemumdicar-
boxylate
135, 107
282, 195, 163,
153, 135, 107
10.0

1
16
G. Leng et al. / Toxicology Letters 107 (1999) 109–121
Fig. 5. Main fragmentations of dimethyl- (CH ), diethyl- (C H ) and dipropyl- (C H ) trans-(E)-chrysanthemumdicarboxylates
3
2
5
3
7
(trans-(E)-CDCA).
clean up steps. The combined organic layers were
vigorously extracted by adding 2 ml 0.5 M
NaOH and the organic layer were discharged
after centrifugation. Again, after washing with
bined and reduced to dryness in a stream of
nitrogen.
The residue was solved in 2 ml of the corre-
sponding alcohol (methanol, ethanol or
propanol). Esterification was accomplished by
adding 0.4 ml concentrated H SO and heating
1.5 ml hexane, the aquateous phase was re-
acidified by adding 0.2 ml concentrated HCl
and extracted twice with 2 ml tert. butyl methyl
ether. The collected organic layers were com-
2
4
(60°C) the solution for 1 h in a water bath. After
cooling, the mixture was diluted with 1 ml of

G. Leng et al. / Toxicology Letters 107 (1999) 109–121
117
water and the derivatives were extracted twice
with 1.5 ml hexane. By this procedure a lot of
matrix interferences were kept back in the aci-
dified alcoholic phase.
The collected organic layers were reduced in a
gentle stream of nitrogen to a volume of 0.5 ml.
An aliquot (2 ml) of the preparation was subjected
to quantitative GC/MS.
Fig. 6. ITD-mass spectra of (A) dimethyl-, (B) diethyl- and (C) dipropyl trans-(E)-chrysanthemumdicarboxylates (full scan 107–282
m/e) obtained by chemical synthesis showing main fragmentations indicated in Fig. 5.

1
18
G. Leng et al. / Toxicology Letters 107 (1999) 109–121
Fig. 7. Total ion chromatogram of the formated CDCA derivatives of standard solutions separated on a DB 5 ms column (standard
solution containing 100 mg/ml of each diastereomere).
3
. Results
tion of CDCA, multiple ion detection (MID) us-
ing up to three characteristical fragment ions with
sufficient high mass-to-charge ratios were
employed.
After the preparation of the synthetical stan-
dards of cis-(E)- and trans-(E)-CDCA, the
racemic mixture was subjected to diastereoselec-
tive preparative HPLC. Characterization of the
separated and purified isomers was by nuclear
magnetic resonance ( H NMR) and derivatization
followed by capillary GC/MS.
Diastereoselective gas chromatography was
achieved for the diethyl and dipropyl esters of
CDCA on a non-polar DB 5 ms capillary column
Aliquotes of standard solutions of the free acid
metabolites and the 2-PBA were added to 2 ml
blank urine samples. For calibration purposes
each of these samples were treated like urine
samples of exposed people. Calibration curves of
cis-(E)- and trans-(E)-CDCA were linear from 10
to 500 mg/l. The purity of the chemicals used was
checked by analysis of blank water samples and
possible background contaminations were recog-
nized by subtracting from the calibration curves.
The limit of detection (LOD) was 20 mg/l for
cis-(E)-CDCA and 10 mg/l for trans-(E)-CDCA
with a signal-to-noise ratio (s/n) higher than 3:1.
The average in-run coefficient of variation of
spiked urine samples was 8% (50 mg/l, n=8).
Internal calibration using 2-phenoxybenzoic
acid (2-PBA, Aldrich) as internal standard was
applied. In addition, no cis-to-trans conversion
was observable and no CDCA was detectable in
blank urine samples of non-exposed people.
In Fig. 8 mass chromatograms of two prepara-
tions of urine samples of a pest control operator
are shown. For the sake of confirmation, two
independent derivative reagents (ethanol and
propanol) were applied. In both cases, only trans-
(E)-CDCA was present in the urine in high yields
1
(
Fig. 7). The major ions and fragmentation path-
ways of the derivatives of CDCA produced by
EI-mass spectrometry were discussed in Figs. 5
and 6. For quantitation, ion fragments showing
the lowest matrix interferences were chosen (Table
1
).
For a biological monitoring, urine samples of a
person exposed to (S)-bioallethrin were subjected
to hydrolytic cleavage. After extraction, CDCA,
the major oxidation product of CA was trans-
formed into appropriate ester derivatives. Separa-
tion
and
characterization
was
by
diastereoselective gas chromatography with elec-
tron impact mass spectrometry (GC/MS). Quanti-
tation was achieved by internal calibration using
2
-PBA as internal standard. Full scan spectrum
mode MS was used for the confirmation of the
metabolites detected in the urine. For quantita-

G. Leng et al. / Toxicology Letters 107 (1999) 109–121
119
(
204916 mg/l) whereas unmetabolized trans-CA
studies in which similar urinary metabolites have
been analyzed after oral administration of (S)-
bioallethrin and related insecticides to rats (Elliott
et al., 1972; Ueda et al., 1975; Brown et al., 1985;
Class et al., 1990).
was only detectable in trace amounts. Moreover,
the concentration of the epimeric isomer cis-(E)-
CDCA was below the limit of detection.
Maximum peak excretion of trans-(E)-CDCA
was within the first 24 h after exposure. In addi-
tion, after 72 h the concentration of the metabo-
lite fell below the limit of detection. The rapid
renal elimination of (S)-bioallethrin is in close
agreement with human exposure studies using the
4
. Discussion
In the present study it was demonstrated that
trans-(E)-CDCA is suitable as a biomarker for
internal (S)-bioallethrin burden. This is in close
agreement with the results of numerous animal
pyrethroid
insecticides
cypermethrin
and
Fig. 8. Mass chromatograms (full scan) of a real 24-h urine sample from a pest control operator exposed to (S)-bioallethrin:
trans-(E)-CDCA (204 mg/l) derivatized as (A) diethyl and (B) dipropyl ester. Major ion fragments and retention times are virtually
identical to those of the corresponding trans-(E)-CDCA standard given in Figs. 6 and 7. Within the limit of detection, the epimeric
cis-(E)-CDCA is not present in the urine.

1
20
G. Leng et al. / Toxicology Letters 107 (1999) 109–121
cyfluthrin. There, peak excretion occurred within
the first 24 h after oral, dermal (Eadsforth and
Baldwin, 1983; Eadsforth et al., 1988; Woollen et
al., 1992; Leng et al., 1997a) or inhalative admin-
istration (Leng et al., 1997b).
Furthermore, in urine samples of persons
not exposed to the insecticides concerned, no
CDCA could be found within the limit of detec-
tion.
In addition, the storage stability of the metabo-
lites is essential for biological monitoring. Storage
experiments demonstrated that trans-(E)-CDCA
is stable for more than a year at −21°C. The
same was shown with metabolites of the halovinyl
type pyrethroid cyfluthrin and cypermethrin
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