Article
J. Agric. Food Chem., Vol. 57, No. 15, 2009 6939
experiments showed that the most rapid isomerization occurred in these
ranges. The samples were capped, covered with aluminum foil, and kept in
the dark at room temperature (20 ( 1 °C). After incubation for 4, 8, 24, and
48 h, duplicate samples were removed and 10 g of anhydrous sodium sulfate
was added to each sample vial to absorb the water. Five milliliters of
methylene chloride was then added, and the sample was mixed on a vortex
for 1 min. The methylene chloride extracts were combined and then filtered
through 10 g of anhydrous sodium sulfate. The combined extract was
evaporated to dryness under a stream of pure nitrogen and redissolved in
0.5 mL of hexane. The stereoisomer composition was analyzed with
GC instrument equipped with a chiral selective column.
Figure 1. Chemical structure of cypermethrin showing chiral positions
(labeled with 1, 3, or R) in the structure.
to light (22), heat (21-23), or organic solvents (13, 18, 22, 24, 25).
However, despite the fact that configurational stability could be an
important consideration in the development of analytical meth-
ods (26) and the interpretation of enantioselective toxicity data (13,
18), abiotic isomerization is often overlooked in chiral pesticides.
In a recent study, we evaluated the configurational stability of
permethrin and CP stereoisomers in a range of polar and nonpolar
solvents, includingmethanol, 2-propanol, acetone, andhexane(24).
Permethrin, which is similar in structure to CP except for the
absence of an R-carbon chiral center, was found to be stable in all
solvents in the dark at room temperature (25 ( 2 °C). In contrast,
CP stereoisomers underwent rapid interconversion in methanol
and 2-propanol, with an enhanced rate in methanol/water mix-
tures. Given that alcohols are among the most widely used solvents
in chemical analysis and bioassays, as well as in pesticide formula-
tions, this study was designed to further explore alcohol-induced
isomerization of CP stereoisomers (1R-cis-RR and 1R-trans-RR)
by considering the influence of alcohol structure (i.e., chain length
and branch chains) and water content. As several other commonly
used pyrethroids (e.g., cyfluthrin, cyhalothrin, and deltamethrin)
share structures similar to that of CP, findings from this study are
expected to have broad implications for the fate and effects of these
compounds in the environment.
GC Analysis. Analysis of stereoisomer composition was conducted on
an Agilent 6890 GC instrument equipped with an electron capture detector
(ECD) (Agilent Technologies, Palo Alto, CA) using a BGB-172 capillary
column [30 m ꢀ 0.25 mm ꢀ 0.25 μm, tert-butyldimethylsilyl-β-cyclodex-
trin dissolved in 15% diphenyl- and 85% dimethylpolysiloxane (BGB
Analytik, Adliwil, Switzerland)]. The detector temperature was 310 °C,
and the makeup gas was nitrogen flowing at a rate of 60 mL/min. The inlet
temperature was 160 °C. The column was initially held at 160 °C for 1 min,
ramped to 230 at a rate of 1 °C/min, and then held at 230 °C for 90 min
until all stereoisomers were eluted. Under the conditions used, all
enantiomeric pairs from the cis diastereomers were well separated, while
the enantiomeric pairs from the trans diastereomers of CP were not
resolved. Preliminary experiments showed that the method detection
limits for the selected stereoisomers were 1-2 ng/mL. Peak areas were
directly used to calculate stereoisomer composition, assuming the same
instrument response for the two stereoisomers.
RESULTS AND DISCUSSION
Recent advances in separation and synthesis techniques have
helped facilitate the routine separation of stereoisomers of a
significant number of chiral compounds for stereoselective envir-
onmental and toxicity assessment (28-30). One complicating
factor in studies related to chiral chemicals is the presence of a
configurationally unstable stereogenic center that is susceptible to
enzymatic and/or nonenzymatic stereoisomer interconversion
(13, 18-20, 22, 24, 25). However, the terms used to define
configurational lability have always been a source of confusion,
particularly in the determination and reporting of rate constants
that refer to the interconversion process. Reist et al. (31) provided a
detailed discussion of semantics and processes involved in config-
urational instabililty. In broad terms, configurational instability
could result in interconversion between enantiomers (defined as
chiral inversion) in compounds with a single element of chirality, or
interconversion between epimers (defined as epimerization) in
compounds that contain two or more elements of chirality (20).
Epimerization of Cypermethrin Stereoisomers in Alcohols. The
enantioselective separation and identification of selected pyre-
throids via GC were described in a previous study (27). The
conversion of CP stereoisomers induced by the heated GC inlet
was relatively small (e3%) under the analytical conditions.
During each sequence of analysis, pure stereoisomers prepared
in n-hexane were included in the analysis to quantify the stereo-
isomer conversion caused by analysis. The calculation of the rate
of epimerization from the various treatments accounted for this
fraction of conversion. A previous study showed that CP stereo-
isomers were relatively stable in n-hexane and methylene chloride
used in the sample preparation (24). In addition, as all samples
were incubated in the dark, the potential contribution from light
to the observed epimerization was considered to be insignificant.
Incubation of 1R-cis-RR-CP in all primary alcohols (ethanol,
methanol, n-propanol, 2-methyl-1-propanol, and n-butanol) re-
sulted in the formation of 1R-cis-RS-CP. Similarly, 1R-trans-RR
epimerized to 1R-trans-RS CP. However, under the analytical
conditions used in this study, the enantiomer pair from the trans
diastereomer (1R-trans-RS and 1S-trans-RR) was not completely
resolved. However, as with the cis stereoisomers, given that
MATERIALS AND METHODS
Chemicals. The analytical standard of CP (98%) was purchased from
Chem Service (West Chester, PA). By using previously established chiral
selective high-performance liquid chromatography (HPLC) methods (27),
individual stereoisomers were resolved and collected for use in further
analysis. Two stereoisomers from two different diastereomers of CP,
1R-cis-RR-CP and 1R-trans-RR-CP, were obtained. The purity of the
isolated stereoisomers was determined to be >99% by HPLC and gas
chromatography (GC) analysis prior to their use. Other solvents or
chemicals used in this study were of analytical or HPLC grade.
Stability of Cypermethrin Stereoisomers in Pure Alcohols. To
understand the dependence of CP stereoisomer stability on alcohol
structures, isomerization was first assessed in a range of alkyl alcohols.
Individual CP stereoisomers were added to 1.0 mL of a solvent in amber
glass GC vials at 10 μg/mL. Epimerization was evaluated in six different
alcohols (methanol, ethanol, n-propanol, 2-methyl-1-propanol, n-butanol,
and 2-butanol). The sample vials were capped with crimp seals, covered
with aluminum foil, and kept in the dark at room temperature (20 ( 1 °C).
After incubation for 8, 24, 48, 96, and 192 h, duplicate samples in separate
vials were removed and solvent evaporated to dryness under nitrogen and
redissolved in 0.5 mL of n-hexane. The samples were analyzed via
GC using a previously developed method to determine the stereoisomer
composition (27).
Stability of Cypermethrin Stereoisomers in Methanol/Water
Mixtures. The second set of experiments was conducted to evaluate
the effect of water in methanol on epimerization of CP stereoisomers.
A 5.0 mL solution of the CP stereoisomer (1R-cis-RR-CP or 1R-trans-RR-
CP) was prepared in a 20 mL glass vial at a concentration of 1.0 μg/mL. The
solution was a mixture of water and methanol at different ratios. Fifteen
water:methanol ratios [100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70,
20:80, 10:90, 8:92, 6:94, 4:96, 2:98, and 0:100 (v/v)] were used, which
represented the water content in methanol ranging from 0 to 100%. Finer
increments (between 0 and 10% water for 1R-trans-RR-CP and between
0 and 2% water for 1R-cis-RR-CP) were also evaluated because preliminary