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Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X
Vol. 2(10), 26-31, October (2012) Res.J.Chem. Sci.
International Science Congress Association 26
Development of A Reversed-Phase High Performance Liquid Chromatographic
Method for Efficient Diastereomeric Separation and Quantification
of Cypermethrin, Resmethrin and Permethrin
Albaseer Saeed S.
Centre for Chemical Sciences and Technology, Institute of Science and Technology, JNT University, Kukatpally, Hyderabad, INDIA
Available online at: www.isca.in
Received 7
th
May 2012, revised 14
th
May 2012, accepted 26
th
June 2012
Abstract
An efficient and simple reversed phase high performance liquid chromatographic method (RP-HPLC) for diastereomeric
separation and quantification of cypermethrin, resmethrin, and permethrin has been developed. Separation was performed
on Phenomenex Luna C18, (4.6 x 150 mm, 5 µm; end capped column). Satisfactory separation of diastereomers was
obtained for the three pyrethroids studied. Good reproducibility of retention time and peak area were achieved. Detection
was performed with UV diode array detector (UV-DAD) at a wavelength of 220 nm. Most peaks were base-separated with Rs
values ranged from 1.6 to 4.5 for most peaks. The optimum mobile phase was composed of a mixture of acetonitrile,
methanol, and water with a mixing ratio of 1:3:1, respectively. The regression coefficients (R
2
) were 0.9991, 0.9951 and
0.9964 with relative standard deviations (RSD%) of 1.95, 2.89 and 1.87, for cypermethrin (CYP), resmethrin (RES) and
permethrin (PER), respectively.
Keywords: Cypermethrin, resmethrin, permethrin, RP-HPLC, pyrethroids, diastereomeric separation.
Introduction
Synthetic pyrethroids (SPs) are potent insecticides and they are
of increasing importance as they have been replacing older
classes of insecticides
1
. Each SP is, however, composed of a
mixture of 2–4 enantiomeric pairs (diastereomers)
2,3
. Several
studies have shown that SPs enantiomers differ significantly in
their biological activities and toxicity
4–7
. In addition, it has
experimentally been proved that SPs are enantioselectively
degraded by microorganisms
8,9
, which affects the distribution
patterns of SPs in the SPs-treated fields
10-12
. Hence,
bioaccumulation of different SPs enantiomers in a particular
area will depend on its content of microorganisms and their
types
13,14
.
As environmental samples are highly complex, it is important
that the analytical method used is capable of providing
reasonable separation of peaks of analytes of interest especially
when such analytes are composed of several diastereomers.
Several HPLC and GC methods have been reported for
quantification of pyrethroids
15,16
. However, most of these
methods require use of expensive speciality columns and
gradient elution with relatively long retention times. Although
spectrometric methods are easy to operate
17
, they are not
suitable for multiresidue analysis, especially with the rapid
growth in the use of hazardous chemicals
18-20
.
Gas chromatography with electron capture detector and normal-
phase high performance liquid chromatography (NP-HPLC) are
the most used analytical techniques for SPs separation.
However, reversed-phase high performance liquid
chromatographic method (RP-HPLC) reported, so far, for the
separation of SPs don not provide efficient diastereomeric
separation. The objective of the work discussed in this paper
was, therefore, to investigate the possibility of developing a
simple, practical and sensitive RP-HPLC method suitable for
accurate separation and quantification of diastereomers of three
SPs, viz., cypermethrin (CYP), resmethrin (RES) and
permethrin (PER) as model SPs. The separation was achieved
on an achiral RP-HPLC column (C
18
). The effects of factors
governing chromatographic separation of SPs diastereomers
were discussed. Further, the effect of the nature of injection
solvent on the peak shape was evaluated by injecting the
analytes in different organic solvents. In addition, the optimized
chromatographic method was further evaluated by analysing
SPs in real water samples.
Material and Methods
Structural Description of Three SPs: Resmethrin’s IUPAC
name is: (5-benzyl-3-furylmethyl (1RS,3RS;1RS,3SR)-2,2-
dimethyl-3-(2-methylpropyl-1-enyl) cyclopropanecarboxylate).
Resmethrin (RES) is a racemic mixture of four isomers: [1R,
trans], [1R, cis], [1S, trans], [1S, cis]. The composition ratio of
the four enantiomers in technical RES is roughly 4:1:4:1. Some
RES isomers have common names, for example, the [1R, cis]-
isomer is called cismethrin and the [1R, trans]- isomer is known
as bioresmethrin. As for biological activities, the [1R, trans]-
isomer possesses the highest insecticidal activity among all RES
isomers followed by the [1R, cis ]- isomer
21
. Permethrin’s
IUPAC name is: (3-phenoxybenzyl (1RS,3RS; 1RS,3SR)-3-(2,2
dichlorovinyl) -2, 2-dimethyl-cyclopropane carboxylate).
Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606X
Vol. 2(10), 26-31, October (2012) Res. J. Chem. Sci.
International Science Congress Association 27
Permethrin (PER) is a racemic mixture of two diastereomers,
and both have low mammalian toxicity. The optical ratio of
1R:1S is 1:1 (racemic). Because the trans isomer is somewhat
less toxic, the (cis:trans) isomeric ratio of 25:75 was chosen for
PER products
22,23
. In addition, it has been shown that [1R, cis]
isomer possesses the highest insecticidal activity among PER
isomers followed by the [1R, trans] isomer
24
.
Cypermethrin’s IUPAC name is: ((RS)-α-cyano-3-
phenoxybenzyl (1RS,3RS;1RS,3SR)-3-(2,2-dichlorovinyl)-2,2-
dimethylcyclo propanecarboxylate). Cypermethrin (CYP)
consists of eight stereoisomers
25
, which form four enantiomeric
pairs (diastereomers), viz., trans (1R-3S-αS + 1S-3R-αR), cis
(1R-3R-αS +1S-3S-αR), trans (1R-3S-αR + 1S-3R-αS), and cis
(1S-3S-αS + 1R-3R-αR). Among the eight isomers, only the 1R-
3R -αS and 1R-3S -αS possess biological activity against pests
and insects
26,27
. The absolute configurations of resmethrin,
permethrin, and cypermethrin are shown in figure-1 with
asterisks indicating asymmetric positions.
Figure-1
Chemical structures of: a) resmethrin, b) permethrin, and c)
cypermethrin. Asymmetric positions are indicated with an
asterisk ‘‘*’’
Materials and Reagents: Permethrin (PER) and Resmethrin
(RES) (PESTANAL
®
, analytical reagent grade, 98.2% and
98.5% purity, respectively) were obtained from Sigma-Aldrich
Co., USA. Cypermethrin (CYP) (technical grade, 95% purity)
was donated by Hyderabad Chemicals Pvt. Ltd, Hyderabad,
India. HPLC grade acetonitrile, acetone, methanol, and n-
hexane were obtained from MERCK (Merck Specialties Pvt.
Ltd, Mumbai, India). Analytical reagent grade sodium chloride
(NaCl) and ethanol were obtained from MERCK (Merck
Specialties Pvt. Ltd, Mumbai, India). Analytical reagent grade
carbon tetrachloride, ethyl acetate, dichloromethane, n-heptane,
toluene, and tetrachloroethane were obtained from RFCL Pvt.
Ltd, New Delhi, India. All reagents were used without further
purification.
Instrumentation: The chromatographic analysis was performed
on a high performance liquid chromatograph (HPLC) (LC-
20AT Prominence, Shimadzu, Japan), equipped with a binary
solvent delivery system, an injection valve with a 20 µ L sample
loop and a UV-diode array detector model SPD-M 20A
Prominence with in-line degasser. The chromatographic
separation was performed on a Phenomenex Luna C
18
, 4.6 x 150
mm, 5 µ m; end capped column), purchased from Phenomenex,
Inc. (411 Madrid Avenue Torrance, CA, USA). Ultrapure water,
purified by a Milli-Q water purification system, Millipore
(Bedford, MA, USA) was used throughout the experiments
unless stated, and was collected on daily basis and degassed
with a vacuum pump and further filtered through 0.45 µ m
membrane (Nylon, Hydrophilic, Millipore, Hyderabad, India).
Samples of 20-µ l volume were injected into HPLC at a mobile
phase flow rate of 1.0 mL min
-1
. A 25-µ L microsyringe
(Hamilton, Switzerland) was used for sample injection into the
HPLC.
Preparation of Reference Solutions: The stock solutions of the
individual standard solutions of the three SPs were prepared in
HPLC-grade acetonitrile (each 10 mg L
-1
) in amber reagent
bottles and kept in the refrigerator at +4
o
C. Working standard
solutions of concentrations ranged from 50-2000 µ gL
-1
were
prepared by dilution of the above stock solutions in HPLC-
grade acetonitrile and were kept in the refrigerator at +4
o
C.
Results and Discussion
Determination of λ
max
of The Tested Pyrethroids: For
efficient chromatographic detection of the tested pyrethroids,
their chromatograms must be recorded at a wavelength at which
they show maximum absorption of UV radiation. The optimum
wavelength (λ
max
) of the three pyrethroids was determined by
recording their UV absorption spectrum using a
spectrophotometer. The overlaid UV-spectra of CYP, RES and
PER are shown in figure-2. For confirmation, the λ
max
was also
evaluated using HPLC by comparing the absorbance of the
investigated pyrethroids at five different wavelengths. The
advantage of recording the spectrum using HPLC is the
possibility of obtaining λ
max
of individual diastereomers of the
three SPs. The results showed that trans forms of permethrin
(PER) diastereomers and resmethrin (RES) diastereomers
exhibited higher UV absorption than their counterparts cis
forms, figure-3.
On the contrary, the cis forms of cypermethrin (CYP)
diastereomers exhibited higher UV absorption than their
counterparts trans forms. For the RP-HPLC analysis, the UV-
detector was set at 220 nm as maximum wavelength (λ
max
) for
simultaneous determination of the three analytes as interfering
peaks observed at wavelengths of 200 nm and 210 nm were
eliminated at this wavelength.
Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606X
Vol. 2(10), 26-31, October (2012) Res. J. Chem. Sci.
International Science Congress Association 28
Figure-2
Overlay of UV-spectra of cypermethrin (CYP), resmethrin
(RES), and permethrin (PER)
Figure-3
Optimization of detection wavelength for the investigated
pyrethroids by HPLC-DAD. Note: for diastereomers’ full
names, please see text
Optimization of Peak Separation: Changes in the composition
of the elution system (mobile phase) will generally affect both
retention factor k, and selectivity factor α, but with less effect on
N (number of column theoretical plates)
28
. In RP-HPLC, the
elution system consists of water as one of the eluent components
and an organic solvent that is usually called a "modifier".
For optimizing the mobile phase composition, several binary
and ternary mixtures of water, acetonitrile (ACN) and methanol
(MeOH) were examined at 220nm as maximum wavelength
(λ
max
) and 1.0 mL/min as a mobile phase flow rate. The
corresponding chromatograms obtained under various mobile
phase compositions are illustrated in figure-4. Starting with
acetonitrile (ACN) as the organic modifier, results showed that
the mobile phase mixture of ACN:H
2
O with a mixing ratio of
4:1, respectively, provides acceptable resolution for both RES
and PER diastereomers, but with very poor diasteriometric
separation of CYP, especially the peaks 1 and 2. So, the peak
pair (peaks 1 and 2) of cypermethrin’s diasteroimers was
considered as the critical band pair. Therefore, for obtaining
acceptable resolution of all diastereomers, acetonitrile was
replaced by methanol. Mixtures of methanol and water with
mixing ratios of 80:20 and 85:15 of methanol and water,
respectively, were able to provide better resolution of all peaks.
Figure-4
Optimization of mobile phase composition for optimum
separation of diastereomers of cypermethrin (CYP),
resmethrin (RES) and permethrin (PER), on an achiral
HPLC column
However, peak resolution of the critical band of CYP
diastereomers was not complete as the second and third peaks
were still, slightly, overlapped. Seeking for optimum peak
resolution, ternary mixtures of acetonitrile (ACN), methanol,
and water were also tested. The results showed that the ACN
content in the ternary mixture of the mobile phase was very
critical. As mobile phase content of ACN increases, there is a
reduction in runtime but, unfortunately, with simultaneous
degradation in separation quality. Acceptable peak separation of
all diasteriomers of the three SPs with reasonable run time was
achieved at a mixture ratio of 20:60:20 of acetonitrile, methanol,
and water, respectively. Most peaks were base-separated with
Rs values ranged from 1.6 to 4.5, except for two band pairs
(band pear of peaks 1&2 and 2&3 of CYP peaks), whose Rs
values were1.0 and 1.1, respectively.
Method Validation: Working standard solutions of
concentrations ranged from 50-2000 µgL
-1
were prepared by
Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606X
Vol. 2(10), 26-31, October (2012) Res. J. Chem. Sci.
International Science Congress Association 29
dilution of the stock solutions in pure HPLC grade acetonitrile.
Triplicate injections of 20 µL injection volume were made for
mixtures of the three compounds at each concentration under
the optimized chromatographic conditions. Peak areas
calculated from the respective chromatograms were plotted
against sample concentration to build calibration curves.
The results showed that the method exhibited linearity in the
tested range of concentrations. The regression coefficients (r
2
)
were 0.9991, 0.9951, and 0.9964 with relative standard
deviations (RSD %) of 1.95, 2.89, and 1.87, for CYP, RES and
PER, respectively. The lowest instrumental detection limit
(LOD) and limit of quantification (LOQ) were determined at a
signal to noise ratio (S/N) of 3 and 10, respectively, and the
results showed that CYP, RES and PER have LOD and LOQ
values ranging from 17–23.4 and 56–78 µg/L, respectively. The
LOD and LOQ values were calculated based on injection of
standard solutions without prior preconcentration. However, as
determination of environmental samples involves a
preconcentration step, which results in obtaining very high
enrichment factors, we can then realize that the LOD and LOQ
values reported here are low enough to allow detecting of these
compounds at trace levels, table-1.
Table-1
Statistical data of calibration curves of investigating
pesticides
Compound Regression
equation* R² RSD
(%)
LOD
(µg/L)
LOQ
(µg/L)
Cypermethrin y = 121.01x
+ 4066.2 0.9991 1.95 22.6 75.2
Resmethrin y = 70.951x
+ 4040.6 0.9951 2.89 17.0 56.7
Permethrin y = 53.664x
+ 4442.1 0.9964 1.87 23.4 78.1
* y = peak area
х = mass of pesticide (µ g)
For the sake of simplifying quantification and peak
identification based on retention time, it is essential that the
variations in values of separation parameters, i.e., k and Rs, for
different analytes being analyzed for, fall within the acceptable
range. The reproducibility of k, N, α, t
R
and Rs through several
runs is given in table-2. The results indicated clearly that the
method provides high degree of precision and reliability. The
retention factor k values ranging from 6.76 to 13.89 fulfil the
condition of acceptable peak separation and analysis time of
0.5< k > 20.
The method intra-day precision (repeatability) of peak areas is
very important for recovery studies based on calculation of peak
areas. For this purpose, six replicates of a concentration within
the linear range were analyzed. The results of repeatability of
peak areas (expressed as relative standard deviation, RSD%)
through several runs for the determination of SPs using the
optimized RP-HPLC showed that the method precision was
within the acceptable range with RSD% <10, except for the
second peak of PER where RSD% was 10.2.
Effect of injection solvent: One of the most problems
encountered during HPLC method development is that the
solvent of standard samples is usually different from the actual
injection sample, which is dictated by the extraction and
preconcentration protocol applied. The injection of sample in a
solvent whose viscosity is different from that of the mobile
phase may cause serious distortions in early eluting bands
29
.
Thus, it was worthy to check the applicability of the developed
chromatographic conditions for different injection solvents. For
this purpose, the three SPs were spiked into aqueous samples,
which were then extracted using different solvents. The
extracting solvents were n-hexane, tetrachloroethane,
dichloromethane, acetonitrile, methanol, and chlorobenzene.
Our results showed that, except some changes in the peaks
retention times, no significant peaks distortion was observed.
These results indicated that the chromatographic conditions
developed are robust and can be used for several injection
solvents.
Table-2
Retention times (t
R
), retention factors (k), separation factors (α) and resolution (Rs) for investigated pesticides under
optimized chromatographic conditions
Compound Peak elution
order
t
R
(min) Retention factor
(k)
Selectivity
factor (α)
Resolution
factor (Rs)
Column Plate
number (N)
x
*
RSD%
x
RSD%
x
RSD%
x
RSD%
x
RSD%
CYP 1 18.40 0.88 6.76 0.99 1.05 0.99 1.6 2.66 6691 1.77
2 19.08 0.84 7.31 0.94 1.04 0.17 1.0 3.96 8059 1.69
3 19.80 0.88 7.89 0.98 1.04 0.07 1.1 2.39 7742 1.77
4 20.90 0.87 8.59 0.96 1.06 0.08 1.6 1.49 7742 1.74
RES 5 22.70 0.83 9.51 0.91 1.10 0.09 2.2 0.93 10181 1.67
6 24.60 0.81 10.47 0.88 1.09 0.23 2.1 2.69 9679 1.62
PER 7 26.96 0.83 11.60 0.90 1.10 0.06 2.1 1.23 9614 1.67
8 32.71 0.97 13.89 1.04 1.23 1.04 4.5 5.22 14148 1.94
* mean value (5 replicates)
Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606X
Vol. 2(10), 26-31, October (2012) Res. J. Chem. Sci.
International Science Congress Association 30
Conclusion
While normal-phase HPLC is usually most suitable for
separating synthetic pyrethroids, the reversed phase-HPLC
method reported by this study has proved its robustness and
efficiency for diastereomeric separation of three SPs. The
sensitivity and accuracy of the method were also assessed.
Mobile phase consisting of ACN and water provides shorter run
time but with poor diastereomeric separation. On the other hand,
mobile phase consisting of MeOH and water provides longer
run time but with good diastereomeric separation of all analytes
except the critical diastereomeric band. A ternary mixture of
ACN, MeOH and water with a mixing ratio of 1:3:1,
respectively, provides the optimum diastereomeric separation of
all analytes. The method exhibited very good robustness and
compatibility with various injection organic solvents, and the
chromatographic separation was achieved at ambient room
temperature.
Acknowledgment
The author thanks Dr. R. Nageswara Rao, Indian Institute of
Chemical Technology (IICT), Hyderabad, India, for fruitful
discussions held with him.
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