Enantiomeric separation of type I and type II pyrethroid insecticides with different chiral stationary phases by reversed-phase high-performance liquid chromatography

Article abstract of DOI:10.1002/chir.22801

The enantiomeric separation of type I (bifenthrin, BF) and type II (lambda-cyhalothrin, LCT) pyrethroid insecticides on Lux Cellulose-1, Lux Cellulose-3, and Chiralpak IC chiral columns was investigated by reversed-phase high-performance liquid chromatography. Methanol/water or acetonitrile/water was used as mobile phase at a flow rate of 0.8?mL/min. The effects of chiral stationary phase, mobile phase composition, column temperature, and thermodynamic parameters on enantiomer separation were carefully studied. Bifenthrin got a partial separation on Lux Cellulose-1 column and baseline separation on Lux Cellulose-3 column, while LCT enantiomers could be completely separated on both Lux Cellulose-1 and Lux Cellulose-3 columns. Chiralpak IC provided no separation ability for both BF and LCT. Retention factor (k) and selectivity factor (α) decreased with the column temperature increasing from 10°C to 40°C for both BF and LCT enantiomers. Thermodynamic parameters including ?H and ?S were also calculated, and the maximum R_s were not always obtained at lowest temperature. Furthermore, the quantitative analysis methods for BF and LCT enantiomers in soil and water were also established. Such results provide a new approach for pyrethroid separation under reversed-phase condition and contribute to environmental risk assessment of pyrethroids at enantiomer level.

Full text of DOI:10.1002/chir.22801

Received: 6 July 2017
Revised: 24 October 2017
Accepted: 22 November 2017
DOI: 10.1002/chir.22801
R E G U L A R A R T I C L E
Enantiomeric separation of type I and type II pyrethroid
insecticides with different chiral stationary phases by
reversed‐phase high‐performance liquid chromatography
1
,2
1,2
1,2
1,2
1,2
1,2
Ping Zhang
| Qian Yu | Xiulong He | Kun Qian | Wei Xiao | Zhifeng Xu
1,2
|
1
,2
Tian Li | Lin He
1
College of Plant Protection, Southwest
Abstract
University, Chongqing, China
2
The enantiomeric separation of type I (bifenthrin, BF) and type II
(lambda‐cyhalothrin, LCT) pyrethroid insecticides on Lux Cellulose‐1, Lux
Cellulose‐3, and Chiralpak IC chiral columns was investigated by reversed‐
phase high‐performance liquid chromatography. Methanol/water or acetoni-
trile/water was used as mobile phase at a flow rate of 0.8 mL/min. The effects
of chiral stationary phase, mobile phase composition, column temperature,
and thermodynamic parameters on enantiomer separation were carefully
studied. Bifenthrin got a partial separation on Lux Cellulose‐1 column and
baseline separation on Lux Cellulose‐3 column, while LCT enantiomers could
be completely separated on both Lux Cellulose‐1 and Lux Cellulose‐3 columns.
Chiralpak IC provided no separation ability for both BF and LCT.
Retention factor (k) and selectivity factor (α) decreased with the column
temperature increasing from 10°C to 40°C for both BF and LCT enantiomers.
Thermodynamic parameters including ΔH and ΔS were also calculated,
Academy of Agricultural Sciences,
Southwest University, Chongqing, China
Correspondence
Lin He, College of Plant Protection,
Southwest University, No. 2 Tiangsheng
Road, Beibei District, Chongqing 400715,
China.
Email: helinok@vip.tom.com
Funding information
Fundamental Research Funds for the
Central Universities, Grant/Award
Number: XDJK2017C067; Chongqing
Postdoctoral Science Foundation, Grant/
Award Number: Xm2017073; China
Postdoctoral Science Foundation, Grant/
Award Number: 2017M612889; National
Natural Science Foundation of China,
Grant/Award Number: 31672085;
and the maximum R were not always obtained at lowest temperature.
s
National Key Research and Development
Program of China, Grant/Award Number:
Furthermore, the quantitative analysis methods for BF and LCT enantiomers
in soil and water were also established. Such results provide a new approach
for pyrethroid separation under reversed‐phase condition and contribute to
environmental risk assessment of pyrethroids at enantiomer level.
2017YFD0200301
KEYWORDS
bifenthrin, enantiomeric separation, enantiomers, HPLC, lambda‐cyhalothrin
1
| INTRODUCTION
usage of pyrethroids has been anticipated to be further
increased with the ban or restriction on organophospho-
rus and organochlorine pesticide in many countries.
3
,4
Pyrethroids are a class of insecticides synthesized based
on natural pyrethrins, the insecticidal compound in cer-
tain species of Chrysanthemum. Considering their out-
standing insecticidal potency and relatively low toxicity,
pyrethroids have been widely used in agriculture and
households over the past several decades. The worldwide
Based on chemical structures, pyrethroids are classified
into type I and type II categories. Type I pyrethroids do
not have α‐cyano group on the phenoxybenzyl moiety,
while type II pyrethroids have α‐cyano group on the
1
2
5
phenoxybenzyl moiety to improve their photostability.
Chirality. 2017;1–12.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

2
ZHANG ET AL.
Structurally, pyrethroids contain 1 to 3 asymmetric
carbon atoms and result in 1 to 4 pairs of enantiomers,
which generally exhibit similar physicochemical and
chemical properties in nonchiral environment and differ-
ent bioactivities in organisms because of different interac-
tion capabilities between enantiomers and naturally
To date, HPLC combined with different types of chiral sta-
tionary phase (CSP) was considered as the most effective
and commonly used method for chiral separation.
2
2
Among the available CSPs, polysaccharide‐based CSPs
including cellulose‐tris‐(3,5‐dimethylphenylcarbamate),
amylose‐tris‐(3,5‐dimethylphenylcarbamate), cellulose‐
tris‐(3,5‐dichlorophenylcarbamate), and cellulose‐tris‐(4‐
methylbenzoate) were most frequently applied in
enantiomer separation. Typically, normal‐phase HPLC is
more widely applied in chiral separation than reversed‐
6-8
chiral biomolecules.
The enantioselective accumula-
tion, degradation, metabolism, and toxicity of pyrethroid
9,10
enantiomers have been widely studied in recent years.
Bifenthrin[BF, 2‐methylbiphenyl‐3‐ylmethyl (1RS,3RS)‐
‐[(Z)‐2‐chloro‐3,3,3‐trifluoroprop‐1‐enyl]‐2,2‐dimethylcyclo-
2
3,24
3
phase HPLC due to better separation efficiency.
propanecarboxylate, 82657‐04‐3] and lambda‐cyhalothrin
LCT, (RS)‐α‐cyano‐3‐phenoxybenzyl (1RS,3RS)‐3‐[(Z)‐2‐
chloro‐3,3,3‐trifluoropropenyl]‐2,2‐dimethylcyclopropane-
carboxylate, 91465‐08‐6] are 2 of the most popular pyrethroids
used to control insect pests and acarids in agriculture
However, reversed‐phase HPLC has been rising fast for
chiral separation in consideration of lower background
signal intensity, easier sample preparation procedures,
and better solubility for polar analytes. Furthermore,
reversed‐phase HPLC is more compatible for
electron spray ionization sources than normal‐phase
[
(Figure 1). In chemical structure, BF and LCT are classi-
25-27
fied into type I and type II pyrethroids, respectively. Com-
mercial BF consists of 2 enantiomers (1S‐cis‐BF and 1R‐
cis‐BF), and LCT only contains 1 of 4 pairs of enantiomers
HPLC in mass spectrometry detection.
For instance,
hexaconazole, epoxiconazole, tebuconazole, myclobutanil,
fenbuconazole, benalaxyl, and metalaxyl were successfully
separated on reversed‐phase HPLC coupled with tandem
([Z]‐1R‐cis‐αS and [Z]‐1S‐cis‐αR). The bioactivities of BF
2
8-31
and LCT enantiomers are significantly different in envi-
ronment and organisms. For example, the LC50 of 1R‐
cis‐BF are 22‐ and 17‐fold more toxic than 1S‐cis‐BF to
mass spectrometry.
Chiral separation of BF and LCT
3
2
enantiomers was conducted by gas chromatography,
14,33
18
HPLC,
capillary electrochromatography, and cyclo-
9,11
Daphnia magna and Ceriodaphnia dubia individually.
dextrin‐modified micellar electrokinetic chromatogra-
34
After BF application, the more toxic enantiomer 1R‐cis‐
phy.
Although a few studies reported the chiral
BF is also more persistent in soil, sediments, and
separation of BF and LCT enantiomers by reversed‐phase
12,13
18,35
water.
For LCT, the toxicity of (−)‐LCT is about 162‐
HPLC,
the complete separation (R > 1.5) was not
s
fold of (+)‐LCT to zebrafish in 96‐hour acute toxicity
achieved under reverse phase condition.
14
test. Therefore, it is necessary to investigate the environ-
mental behaviors of chiral pyrethroid pesticide at enantio-
mer level.
In the context of this study, BF and LCT enantiomers
were successfully separated on Lux Cellulose‐1, Lux
Cellulose‐3, and Chiralpak IC chiral columns. The effects
of CSPs, mobile phase composition, column temperature,
and thermodynamic parameters on resolution were care-
fully evaluated. Chromatographic parameters including
retention factor (k), selectivity factor (α), and resolution
Enantiomeric separation of chiral pesticide enantio-
mers is a key basis for enantiomer‐specific behavior
research. Generally, reversed‐phase high‐performance
1
5
16
liquid chromatography (HPLC), normal‐phase HPLC,
17
18
gas chromatography, capillary electrophoresis, ultra‐
performance convergence chromatography, cyclodex-
trin‐modified micellar electrokinetic chromatography,
supercritical fluid chromatography, etc. have been
factor (R ) were employed to evaluate the separation effi-
s
19
ciency. Furthermore, the quantitative analysis methods
for BF and LCT enantiomers in soil and water were also
established. Such results shed new light on chiral separa-
tion of type I and type II pyrethroids and contribute to
environmental risk assessment of pyrethroids at enantio-
mer level.
20
21
developed to separate enantiomers of chiral pesticides.
2
| MATERIALS AND METHODS
.1 | Chemicals and reagents
2
Bifenthrin (purity ≥ 97.0%) and LCT (purity ≥ 98.0%)
were purchased from J&K Scientific Co. Ltd. (Beijing,
China). Stock solutions of BF and LCT were prepared
with acetonitrile and stored at 4°C. Water was purified
with a Millipore Milli‐Q system (Billerica, USA).
FIGURE 1 Chemical structure of bifenthrin (BF) and lambda‐
cyhalothrin (LCT). *Asymmetric carbon atom

ZHANG ET AL.
3
Acetonitrile and methanol (HPLC grade) were purchased
from Fisher scientific (Beijing, China). All other regents
were analytical grade and purchased from Sinopharm
Chemical Reagent Co. Ltd. (Beijing, China).
of solvent A (methanol or acetonitrile) and solvent B
(water). In each run, the flow rate was 0.8 mL/min, and
the 20‐μL sample was injected with detection wavelength
at 220 nm. Column temperature was changed from 10°C
to 40°C. The retention factor (k = [t − t ]/t ), separation
0
0
factor (α = k /k ), and resolution factor (R = 2[t ‐t ]/
2
1
s
2 1
2.2 | Apparatus
[
w + w ]) were calculated.
1 2
Reversed‐phase chiral HPLC separations were conducted
on an Agilent 1260 series HPLC system (Santa Clare,
USA), equipped with G1322 degasser, G1311B quatpump,
G1329B autosampler with a 100‐μL sample loop, G1316A
column compartment, and G1315D diode array detector.
The signal was collected and analyzed by an Agilent
workstation.
2
.4 | Extraction of bifenthrin and lambda‐
cyhalothrin enantiomers from soil and
water
−
1
Three spiked levels (0.02, 0.5, and 5 mg kg ) of BF and
LCT standard solution were added to soil (5 g, dried at
room temperature), respectively. After that, 25‐mL aceto-
nitrile, 2‐g anhydrous sodium sulfate, and 1‐g sodium
chloride were added to 50‐mL polypropylene centrifuge
tubes. The mixture was shaken in rotary vibrator for
10 minutes at 300 rpm, exposed to ultrasonic vibration
for 15 minutes, and then centrifuged at 3500 rpm for
5 minutes. The extraction solvent was filtered through
10 g of anhydrous sodium sulfate for dehydration. The
2.3 | Chromatographic conditions
Enantiomers of BF and LCT were separated on Lux
Cellulose‐1 (250 mm × 4.6 mm [i.d.], 5 μm), Lux
Cellulose‐3 (250 mm × 4.6 mm [i.d.], 5 μm), and
Chiralpak IC (250 mm × 4.6 mm [i.d.], 5 μm) chiral
columns, respectively. The mobile phase was composed
FIGURE 2 Chiral resolution
chromatograms of bifenthrin (BF) on Lux
Cellulose‐3 (ACN/H
0/20, and D 70/30), Lux Cellulose‐1
ACN/H O, E 75/25, F 70/30, G 65/35, and
H 60/40), and lambda‐cyhalothrin (LCT)
on Lux Cellulose‐3 (ACN/H O, H 80/20,
I 70/30, J 60/40, and K 50/50) and Lux
Cellulose‐3 (MEOH/H O, L 100/0, M 95/5,
N 90/10, and O 85/15)
2
O, A 95/5, B 90/10, C
8
(
2
2
2

4
ZHANG ET AL.
residue soil was extracted by another 25‐mL acetonitrile,
and all the extraction steps were the same as previous
procedures. As for water sample, 25‐mL ethyl acetate
and 1‐g sodium chloride were added, and other steps
were the same as soil. The filtered solvent was
combined into a pear‐bottomed flask and evaporated to
near dryness by using a rotary vacuum evaporator at
40°C and reconstituted in 1.0‐mL acetonitrile for chiral
HPLC analysis.
2.5 | Method validation
The performance of developed method was evaluated
based on parameters including linearity, accuracy,
TABLE 1 Enantiomeric separation results of bifenthrin (BF) and lambda‐cyhalothrin (LCT) enantiomers on 3 chiral columns using
MEOH/H O or ACN/H O as mobile phase
2
2
Compound
Mobile Phase
Ratio (vol/vol)
k1
2.08
5.08
k2
2.08
5.15
α
Rs
0.00
0.24
0.25
BF
Lux Cellulose‐1 MEOH/H
2
O
95/5
1.00
1.01
1.01
9
8
0/10
5/15
13.47
13.67
BF
Lux Cellulose‐1 ACN/H
2
O
90/10
1.01
3.88
6.15
10.51
17.98
1.01
3.98
6.31
10.80
18.47
1.00
1.02
1.03
1.03
1.03
0.00
0.45
0.47
0.50
0.54
7
7
6
6
5/25
0/30
5/35
0/40
BF
BF
Lux Cellulose‐3 MEOH/H
2
O
100/0
0.59
1.60
4.14
1.68
4.95
13.53
2.86
3.09
3.26
6.18
8.88
8.91
9
9
5/5
0/10
Lux Cellulose‐3 ACN/H
2
O
100/0
0.24
0.15
0.14
0.53
1.89
4.99
0.34
0.33
0.47
1.55
5.14
13.97
1.40
2.25
3.29
2.93
2.73
2.80
1.34
2.47
3.49
7.64
10.08
13.30
9
9
8
7
5
5/5
0/10
0/20
0/30
5/45
BF
Chiralpak IC MEOH/H
2
O
90/10
0/20
0.68
4.10
0.68
4.10
1.00
1.00
—
—
8
BF
Chiralpak IC ACN/H
2
O
90/10
0/40
0.23
5.65
0.23
5.65
1.00
1.00
—
—
6
LCT
Lux Cellulose‐1 MEOH/H
2
O
100/0
0.50
1.14
2.91
7.39
0.55
1.32
3.43
8.84
1.10
1.16
1.18
1.20
0.36
1.02
1.47
2.03
9
9
8
5/5
0/10
5/15
LCT
Lux Cellulose‐1 ACN/H
2
O
100/0
0.22
0.63
2.05
6.00
18.41
0.24
0.70
2.26
6.64
20.44
1.10
1.11
1.10
1.11
1.11
0.44
0.80
1.26
1.61
1.96
9
8
7
6
0/10
0/20
0/30
0/40
LCT
LCT
Lux Cellulose‐3 MEOH/H
2
O
100/0
0.30
0.77
2.07
5.98
0.69
1.96
5.79
2.31
2.54
2.79
3.04
4.60
4.27
6.47
8.29
9
9
8
5/5
0/10
5/15
18.22
Lux Cellulose‐3 ACN/H
2
O
80/20
0.42
1.25
3.14
0.48
1.41
3.58
1.13
1.13
1.14
1.15
0.82
1.28
1.53
2.26
7
6
5
0/30
0/40
0/50
14.76
17.02
LCT
LCT
Chiralpak IC MEOH/H
2
O
90/10
0/20
1.24
6.59
1.24
6.59
1.00
1.00
—
—
8
Chiralpak IC ACN/H
2
O
70/30
0/40
2.03
5.27
2.03
5.27
1.00
1.00
—
—
6

ZHANG ET AL.
5
precision, limit of detection (LOD), and limit of quantita-
tion (LOQ). The stock standard solutions were prepared
in acetonitrile and diluted to a series of concentrations
Cellulose‐3, respectively. As for LCT, the baseline separa-
tions were achieved on both Lux Cellulose‐1 and Lux
Cellulose‐3, and maximum R were 2.03 and 8.29 at
s
−
1
(0.05, 0.1, 0.5, 1, 2, 5, and 10 mg L ). Linearity was
MEOH/H O ratio of 85/15, individually. When ACN/
2
obtained by linear regression of peak area of enantiomer
versus the injected concentration. The LOD for each
enantiomer was defined as the concentration that pro-
duced a signal‐to‐noise ratio of 3, while the LOQ was con-
sidered as the lowest concentration in calibration curve
with acceptable accuracy and precision. The recovery
and relative standard deviation (RSD) of triple replicates
at 3 spiked concentrations (0.02, 0.5, and 5 mg kg ) were
employed to evaluate the accuracy and precision of the
developed method. The stability of BF and LCT stock
solution was checked weekly and indicated that those 2
compounds were stable under −20°C condition.
H O was used as mobile phase, BF got partial separation
2
on Lux Cellulose‐1 with maximum R = 0.54 and com-
s
plete separation on Lux Cellulose‐3 with maximum
Rs = 13.30, individually. Lambda‐cyhalothrin enantio-
mers were completely separated on Lux Cellulose‐1 and
Lux Cellulose‐3 columns. The maximum R is 1.96 on
s
Lux Cellulose‐1 with mobile phase ratio of 60/40 and
2.26 on Lux Cellulose‐3 with ratio of 50/50, respectively.
Chiralpak IC had no ability to separate both BF and LCT
enantiomers when MEOH/H O or ACN/H O was used
−
1
2
2
as mobile phase.
The separation efficiencies of MEOH/H O and ACN/
2
H O on Lux Cellulose‐1 for both BF and LCT were almost
2
the same. However, MEOH/H O exhibited better separa-
2
3
3
| RESULTS AND DISCUSSION
.1 | Chiral resolutions
tion ability than ACN/H O on Lux Cellulose‐3 for BF,
2
while ACN/H O was better for LCT on Lux Cellulose‐3,
2
which indicated that methanol and acetonitrile exhibited
different separation capacities on the same column and
the separation efficiencies were related not only to mobile
phase but also CSP. It is reported that hydrogen bonding,
π‐π, and dipole‐dipole interactions were main forces for
The enantiomeric separations of BF and LCT enantio-
mers on 3 chiral columns were performed under
reversed‐phase HPLC condition (Figure 2). Methanol/
water (MEOH/H O) or acetonitrile/water (ACN/H O)
was used as mobile phase to optimize chromatographic
condition at a flow rate of 0.8 mL/min at 20°C. The
separation parameters including retention factors (k1
and k ), separation factor (α), and resolution factor (R )
were summarized in Table 1, and R > 1.5 was consid-
ered as complete separation. When MEOH/H O was
used as mobile phase, BF enantiomers got a partial sepa-
ration (maximum R = 0.25) and complete separation
2
2
36
the chiral resolution. In our study, methanol is a pro-
tonic solvent, which could donate and accept proton in
hydrogen‐bond formation. However, acetonitrile is just a
weak proton acceptor in the process of hydrogen‐bond
formation. Therefore, the different separation efficiencies
between methanol and acetonitrile may be caused by
the different hydrogen‐bond interaction between mobile
phase and analyte. In general, a lower ratio of methanol
or acetonitrile in mobile phase leads to a higher resolution
2
s
s
2
s
(maximum Rs = 8.91) on Lux Cellulose‐1 and Lux
FIGURE 3 The effects of temperature
on lambda‐cyhalothrin (LCT) enantiomer
separation with Lux cellulose‐1 column
(
ACN/H
0°C, and D 40°C) and Lux cellulose‐1
column (MEOH/H O [95/5], E 10°C,
F 20°C, G 30°C, and H 40°C)
2
O [70/30], A 10°C, B 20°C, C
3
2

6
ZHANG ET AL.
TABLE 2 Effects of temperature on bifenthrin (BF) and lambda‐cyhalothrin (LCT) separation with Lux Cellulose‐1 and Lux Cellulose‐3
chiral columns
Mobile Phase
(vol/vol)
Temperature
(°C)
Compound
k1
k2
α
Rs
BF
Lux Cellulose‐1
MEOH/H O (90/10)
2
10
15
5.54
5.08
4.32
4.05
3.69
3.34
3.04
5.65
5.17
4.38
4.05
3.69
3.34
3.04
1.02
1.02
1.01
1.00
1.00
1.00
1.00
0.32
0.27
0.21
0.00
0.00
0.00
0.00
2
2
3
3
4
0
5
0
5
0
BF
Lux Cellulose‐1
ACN/H O (70/30)
10
15
4.72
4.61
4.49
4.42
4.26
4.09
3.91
4.88
4.76
4.62
4.54
4.37
4.19
3.99
1.03
1.03
1.03
1.03
1.03
1.02
1.02
0.92
0.56
0.54
0.52
0.47
0.46
0.45
2
2
2
3
3
4
0
5
0
5
0
BF
Lux Cellulose‐3
MEOH/H O (95/5)
10
15
1.77
1.52
1.60
1.37
1.38
1.26
1.14
6.01
4.89
4.95
3.95
3.82
3.33
2.84
3.39
3.22
3.09
2.89
2.78
2.63
2.49
8.02
8.01
8.32
8.45
8.60
8.42
8.25
2
2
2
3
3
4
0
5
0
5
0
BF
Lux Cellulose‐3
ACN/H O (80/20)
10
15
0.56
0.55
0.52
0.49
0.47
0.45
0.41
1.84
1.72
1.58
1.41
1.26
1.12
0.98
3.29
3.13
3.05
2.85
2.67
2.51
2.37
7.82
7.71
7.63
7.52
7.30
6.42
6.10
2
2
2
3
3
4
0
5
0
5
0
LCT
LCT
LCT
Lux Cellulose‐1
MEOH/H O (85/15)
10
15
3.96
3.51
2.91
2.80
2.50
2.25
2.06
4.79
4.20
3.43
3.28
2.90
2.59
2.35
1.21
1.20
1.18
1.17
1.16
1.15
1.14
1.63
1.60
1.47
1.29
1.25
1.17
1.09
2
2
2
3
3
4
0
5
0
5
0
Lux Cellulose‐1
ACN/H O (70/30)
10
15
6.53
6.23
6.00
5.65
5.32
4.99
4.80
7.34
6.95
6.64
6.24
5.85
5.47
5.22
1.12
1.12
1.11
1.11
1.10
1.09
1.09
1.78
1.75
1.61
1.59
1.45
1.35
1.33
2
2
2
3
3
4
0
5
0
5
0
Lux Cellulose‐3
MEOH/H O (95/5)
10
15
1.14
1.01
0.90
0.81
0.72
0.64
0.58
3.35
2.76
2.39
2.00
1.64
1.38
1.19
2.95
2.75
2.66
2.48
2.27
2.16
2.05
3.86
4.64
4.27
4.02
4.37
4.64
4.72
2
2
2
3
3
4
0
5
0
5
0
(Continues)

ZHANG ET AL.
7
TABLE 2 (Continued)
Mobile Phase
(vol/vol)
Temperature
(°C)
Compound
k1
k2
α
Rs
LCT
Lux Cellulose‐3
ACN/H O (60/40)
2
10
15
3.50
3.45
3.32
3.23
3.10
2.96
2.80
3.98
3.94
3.78
3.68
3.53
3.36
3.18
1.14
1.14
1.14
1.14
1.14
1.14
1.13
1.12
1.53
1.46
1.55
1.56
1.58
1.52
2
2
3
3
4
0
5
0
5
0
and longer elution time in enantiomer separation under
reversed‐phase HPLC. In accordance with this phenome-
non, the retention factors (k) and resolution factor (Rs)
values decreased with increasing ratio of methanol and
acetonitrile in mobile phase. Previous study reported par-
tial separation of LCT enantiomers on Chiralcel OD‐R
decreased from 1.78 to1.33, 6.53 to 4.80, and 7.34 to 5.22,
respectively, on Lux Cellulose‐1 column with ACN/H O
2
ratio of 70/30. However, temperature sometimes has little
effects on chiral resolution such as the separation of BF
enantiomers on Lux Cellulose‐3 with MEOH/H O ratio
2
of 95/5 and the maximum R achieved at 30°C. Similar
s
chiral column with maximum R = 1.17 under reversed‐
phase HPLC. In the present study, the complete separa-
results were also obtained from chiral separation of LCT
s
30
enantiomers on Lux Cellulose‐3 (MEOH/H O [95/5])
2
tion (R > 1.5) of BF and LCT enantiomers was achieved
and the maximum R = 4.72 obtained at 40°C.
s
s
on both Lux Cellulose‐1 and Lux Cellulose‐3 column by
using MEOH/H O and ACN/H O as mobile phase. The
2
2
3.3 | Thermodynamic parameters
maximum R were 13.30 and 8.29 for BF and LCT, respec-
s
tively. Thus, this study provided a baseline separation of
BF and LCT enantiomers under reversed‐phase HPLC.
To understand the thermodynamic driving forces
involved in BF and LCT enantiomer separation, Van't
Hoff equations were employed to calculate the standard
enthalpy (ΔH) and entropy (ΔS) according to retention
factors (k) and selectivity factor (α) obtained from differ-
ent temperatures (Figure 4).
3.2 | Influences of temperature
Column temperature is an important factor for chiral res-
olution and contributes to understanding the mechanism
of chiral recognition. In this study, the influences of
column temperature on BF and LCT enantiomer separa-
tion were carefully investigated from 10°C to 40°C with
increment of 5°C on Lux Cellulose‐1 and Lux Cellulose‐
ΔH ΔS
In k ¼ − RT
þ
þ Inφ
(1)
R
ΔΔH ΔΔS
In a ¼ − RT
3
columns (Figure 3). Table 2 summarized chromato-
þ
(2)
graphic conditions and separation results of temperature
study. In BF enantiomer separation, the components of
mobile phase were MEOH/H O (90/10) and ACN/H O
R
2
2
(
(
70/30) for Lux Cellulose‐1, as well as MEOH/H O
2
95/5) and ACN/H O (80/20) for Lux Cellulose‐3. As for
2
LCT, MEOH/H O (85/15) and ACN/H O (70/30) were
2
2
used as mobile phase on Lux Cellulose‐1, as well as
MEOH/H O (95/5) and ACN/H O (60/40) on Lux
2
2
Cellulose‐3 in consideration of eluted time and separation
efficiency. The results indicated that temperature could
significantly affect the interaction between 2 pyrethroid
enantiomers and CSP. In general, lower temperature
leads to longer eluted time, wider peak, and higher resolu-
tion. In our study, resolution factor (R ) and retention
s
factor (k , k ) decreased with increasing temperature for
1
2
BF and LCT enantiomers on Lux Cellulose‐1. For
instance, the R , k , and k values of LCT enantiomers
FIGURE 4 Van't Hoff plots of LCT on Lux Cellulose‐1 with
ACN/H O ratio of 70/30
s
1
2
2

8
ZHANG ET AL.

ZHANG ET AL.
9
where R presents the gas constant (8.314 J/[mol K]), T
Table 3 summarized the thermodynamic parameters
is the absolute temperature, and φ is the phase ratio. ΔH
and ΔS are the molecular enthalpy and entropy involved
in enantiomer separation. ΔΔH represents the value of
ΔH ‐ΔH , and ΔΔS is the value of ΔS ‐ΔS . Based on
Equation 1, the values of −ΔH/R and (ΔS/R + lnφ) could
be calculated from the slop and intercept according to
linear regression of lnk versus 1/T. Similarly, the values
of −ΔΔH/R and ΔΔS/R could also be calculated based
on linear regression of lnα versus 1/T.
of BF and LCT enantiomers on Lux Cellulose‐1 and Lux
Cellulose‐3 chiral columns by using MEOH/H O and
2
ACN/H O as mobile phase. The ΔH values of BF and
2
LCT enantiomers range from −4.51 to −17.24 kJ/mol
and −5.47 to −25.67 kJ/mol, respectively. The negative
values of ΔH clearly indicate that the transformation of
BF and LCT enantiomers from mobile phase to CSP is
mainly driven by enthalpy. The ΔΔH of BF and LCT
enantiomers on 2 types chiral columns under specified
2
1
2
1
FIGURE 5 Representative
chromatograms of bifenthrin (BF; Lux
Cellulose‐3, MEOH/H
lambda‐cyhalothrin (LCT; Lux Cellulose‐
, MEOH/H O = 90/10) enantiomers
2
O = 95/5) and
3
2
extracted from soil and water. (A) BF
standard solution; (B) BF extracted from
−
1
water at 5 mg L spiked level; (C) BF
−
1
extracted from soil at 5 mg kg spiked
level; (D) LCT standard solution; (E) LCT
−
1
extracted from water at 5 mg L spiked
level; (F) LCT extracted from soil at
−1
5
mg kg spiked level

10
ZHANG ET AL.
mobile phase condition range from −0.24 to −8.19 kJ/mol
and −0.13 to −9.04 kJ/, individually, which means the
ΔH values of second eluted enantiomers are more nega-
tive than the first eluted enantiomer, implying that the
CSPs have stronger interaction with the second eluted
enantiomers than the first one. Moreover, the negative
values of ΔΔH indicate that lower temperature leads to
better separation of these 2 chiral pyrethroid enantiomers
on Lux Cellulose‐1 and Lux Cellulose‐3 columns.
90.83 to 100.30% from soil and 92.64 to 98.48% from water
with RSD less than 7% (Table 4). The LODs for BF and
−
1
LCT enantiomers were 0.01 and 0.015 mg L , respec-
−
1
tively, and the corresponding LOQ is 0.05 mg L for both
BF and LCT based on the lowest concentration in calibra-
tion curve with acceptable RSD < 20%. Such results
indicated that the methods were robust and reliable for
residual analysis of BF and LCT enantiomers in environ-
mental samples.
2
However, the correlation coefficient (R ) of linear
regression of lnα versus 1/T is 0.623 on Lux Cellulose‐3
with ACN/H O ratio of 60/40, which indicates that
multiple interaction forces exist between LCT enantio-
mers and CSP.
2
4 | CONCLUSION
In the present study, type I (BF) and type II (LCT) pyre-
throid enantiomers were separated on Lux Cellulose‐1,
Lux Cellulose‐3, and Chiralpak IC chiral columns by
reversed‐phase high‐performance liquid chromatography.
Bifenthrin gets a baseline separation on Lux Cellulose‐3
and partial separation on Lux Cellulose‐1 with maximum
R = 13.30 and R = 0.54, respectively, while LCT enantio-
3
.4 | Bifenthrin and lambda‐cyhalothrin
enantiomer analysis in environmental
samples
The developed chiral HPLC method was validated for the
quantitative analysis of BF and LCT enantiomers in soil
and water (Figure 5). The calibration curves obtained
s
s
mers could be completely separated on Lux Cellulose‐1
and Lux Cellulose‐3 with maximum R = 2.03 and
s
−
1
from the concentration range of 0.05 to 10 mg L showed
Rs = 8.29, individually. Chiralpak IC provides no separa-
good linearity for the first eluted BF enantiomer
tion ability for both BF and LCT. ACN/H O exhibits
2
2
(
y = 38.391x + 1.2195, R = 0.9984), second eluted BF
better separation efficiency than MEOH/H O on Lux
2
2
enantiomer (y = 42.428x + 0.6537, R = 0.9999), first
eluted LCT enantiomer (y 34.188x 2.7852,
= 0.9998), and second eluted LCT enantiomer
Cellulose‐1 and Lux Cellulose‐3 chiral columns for BF,
=
+
whereas MEOH/H O is much better than ACN/H O on
2
2
2
R
Lux Cellulose‐3, and these 2 mobile phases exhibit similar
separation potency on Lux Cellulose‐1 for LCT. Retention
factor (k) and selectivity factor (α) decreased with the
column temperature increasing from 10°C to 40°C for
both BF and LCT. Furthermore, the quantitative
analysis methods for BF and LCT enantiomers in soil
2
(
y = 29.717x + 2.1693, R = 1), respectively. Average
recoveries of BF enantiomers at 3 spiked levels were
1.42 to 99.74% from soil and 90.82 to 101.50% from water
9
with the RSD ranging of 2.54 to 5.54% and 3.29 to 5.69%,
respectively. The recoveries of LCT enantiomers were
TABLE 4 Recovery and precision of bifenthrin (BF) and lambda‐cyhalothrin (LCT) enantiomers from soil and water (n = 3)
E1 (First Eluted Enantiomer) E2 (Second Eluted Enantiomer)
Spiked Levels
(mg kg or mg L
−
1
−1
Sample
BF
)
Recovery (%)
RSD
Recovery (%)
RSD
Soil
0.02
98.67 ± 4.25
95.63 ± 5.30
96.15 ± 3.51
93.35 ± 5.08
101.50 ± 3.75
90.82 ± 5.16
4.31
5.54
3.65
5.44
3.70
5.69
99.74 ± 5.01
91.42 ± 2.33
97.42 ± 3.78
95.69 ± 5.12
93.31 ± 4.43
94.45 ± 3.11
5.03
0
5
.5
2.54
3.88
5.35
4.74
3.29
Water
0.02
0
5
.5
LCT
Soil
0.02
100.30 ± 4.59
97.25 ± 4.15
99.76 ± 3.70
97.35 ± 6.50
93.67 ± 4.81
96.80 ± 4.52
4.58
4.27
3.71
6.68
5.13
4.67
98.69 ± 5.34
96.56 ± 4.76
90.83 ± 3.99
93.06 ± 4.43
98.48 ± 4.73
92.64 ± 4.63
5.42
4.93
4.40
4.76
4.80
5.00
0
5
.5
Water
0.02
0
5
.5

ZHANG ET AL.
11
and water were also established. Such results provide a
new approach for pyrethroid separation under reversed‐
phase condition and contribute to risk assessment of
pyrethroids at enantiomer level.
9. Ye J, Zhao M, Niu L, Liu W. Enantioselective environmental
toxicology of chiral pesticides. Chem Res Toxicol.
2015;28(3):325‐338.
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ACKNOWLEDGMENTS
1. Liu W, Gan J, Schlenk D, Jury WA. Enantioselectivity in
environmental safety of current chiral insecticides. Proc Natl
Acad Sci U S A. 2005;102(3):701‐706.
We gratefully acknowledge the financial support from the
National Key Research and Development Program of
China (2017YFD0200301), National Natural Science
Foundation of China (31672085), China Postdoctoral
2. Li Z, Zhang Z, Zhang L, Leng L. Isomer‐ and enantioselective
degradation and chiral stability of fenpropathrin and fenvalerate
in soils. Chemosphere. 2009;76(4):509‐516.
Science
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Chongqing
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risk assessment of modern chiral pesticides. Environ Pollut.
Postdoctoral Science Foundation (Xm2017073), and
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CONFLICT OF INTEREST STATEMENT
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separation of chiral pesticides by liquid chromatography on
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The authors declare no competing financial interest.
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resolution of chiral pesticides by high‐performance liquid
chromatography. J Agric Food Chem. 2006;54(5):1577‐1583.
Ping Zhang http://orcid.org/0000-0001-9661-8267
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