Validation of a Chiral Liquid Chromatographic Method for the Degradation Behavior of Flumequine Enantiomers in Mariculture Pond Water

Article abstract of DOI:10.1002/chir.22625

In this work, flumequine (FLU) enantiomers were separated using a Chiralpak OD-H column, with n-hexane-ethanol (20:80, v/v) as the mobile phase at a flow rate of 0.6 mL/min. Solid phase extraction (SPE) was used for cleanup and enrichment. The limit of detection, limit of quantitation, linearity, precision, and intra/interday variation of the chiral high-performance liquid chromatography (HPLC) method were determined. The developed method was then applied to investigate the degradation behavior of FLU enantiomers in mariculture pond water samples. The results showed that the degradation of FLU enantiomers under natural, sterile, or dark conditions was not enantioselective. Chirality 28:649–655, 2016.

Full text of DOI:10.1002/chir.22625

CHIRALITY (2016)
Validation of a Chiral Liquid Chromatographic Method for the
Degradation Behavior of Flumequine Enantiomers in Mariculture
Pond Water
*
*
YAN-FEI WANG, XIAO-FENG GAO, HUO-XI JIN, YANG-GUANG WANG, WEI-JIAN WU AND XIAO-KUN OUYANG
School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan, P.R. China
ABSTRACT
In this work, flumequine (FLU) enantiomers were separated using a Chiralpak
OD-H column, with n-hexane-ethanol (20:80, v/v) as the mobile phase at a flow rate of 0.6
mL/min. Solid phase extraction (SPE) was used for cleanup and enrichment. The limit of detec-
tion, limit of quantitation, linearity, precision, and intra/interday variation of the chiral high-
performance liquid chromatography (HPLC) method were determined. The developed method
was then applied to investigate the degradation behavior of FLU enantiomers in mariculture
pond water samples. The results showed that the degradation of FLU enantiomers under natural,
sterile, or dark conditions was not enantioselective. Chirality 00:000–000, 2016. © 2016 Wiley Pe-
riodicals, Inc.
KEY WORDS: flumequine enantiomer; chiral analysis; enantioselective behavior; mariculture
pond water
Flumequine (FLU)1 exhibits broad-spectrum antimicrobial
activities and is a quinolone-containing antibiotic widely used
for the prevention and treatment of a variety of infectious dis-
eases in aquaculture.2–4 However, FLU exhibits low bioavail-
ability in aquatic species.5–7 Due to the frequent use of FLU
in mariculture farms, residual FLU may remain in water. This
issue has garnered significant attention because residual FLU
may result in the accumulation of FLU in fish, which is poten-
tially harmful to humans. Furthermore, the emergence of
antibiotic-resistant bacteria8 has been attributed to the pres-
ence of antibiotics in water.9
Enantiomers of some chiral compounds, including antibi-
otics, exhibit different biological activities including toxicity
and effects on organisms and the environment.10–13 The degra-
dation and metabolic products of such compounds may also be
chiral and typically differ in their chemical and toxicological
properties, as compared to the parent compounds. As such,
they may exhibit various ecological risks.14–16 Notably, FLU
contains an asymmetric carbon center and has two enantio-
mers (shown in Fig. 1). As FLU is chiral, it might exhibit the
aforementioned issues. Previous studies have suggested that
the enantiomers of FLU exhibit significant differences with re-
gard to their antimicrobial effectiveness.17 While environmen-
tal factors such as light18 and microbial activities19 would affect
the degradation of rac-FLU, these factors may also result in dif-
ferent degradation behaviors of FLU enantiomers. Further-
more, FLU is usually employed in the racemic form in
aquaculture farms; therefore, traditional risk evaluations of
FLU are insufficient and elucidating the degradation behavior
of FLU enantiomers in water is necessary.
under natural, sterile, and dark conditions using the developed
method. Since the absolute configuration of enantiomers is an
important parameter for understanding the biochemical prop-
erties of chiral compounds,20 the absolute configurations of
FLU enantiomers were determined by comparing the experi-
mental and calculated electronic circular dichroism (ECD)
spectra.11,13
MATERIALS AND METHODS
Materials
rac-FLU (>98%) was purchased from Aladdin Reagent (Shanghai,
China). FLU enantiomers standards (>98%) were obtained from Daicel
Chiral Technologies (Tokyo, Japan) (Shanghai, China). HPLC-grade etha-
nol and n-hexane were purchased from Merck (Darmstadt, Germany). De-
ionized water was obtained using a Milli-Q water purification system from
Millipore (Molsheim, France). Acetic acid (>99.0%), ammonium acetate
(>99.0%), and methanol (>99.8%) were purchased from Aladdin Reagent.
A chiral analytical column (Chiralpak OD-H, 4.6 mm × 250 mm i.d., 5
μm) was purchased from Daicel.
ECD Spectroscopy
ECD spectroscopy was carried out using a Jasco-J815 spectropolarime-
ter (Tokyo, Japan) at 25°C. The FLU enantiomers (>98%) were dissolved
in methanol at a concentration of 125 μg/mL. The ECD spectra were col-
lected at a wavelength range of 200 to 500 nm at a scan speed of 100
nm/min. A quartz cell with a pathlength of 1 mm was used, and the aver-
age spectrum from three scans is reported.
Calculated ECD spectra were obtained using time-dependent density
functional theory (TDDFT)21 employing the B3LYP functional using
Gaussian 09,13 and were analyzed using GUIs GaussView (v. 5.0). The ini-
tial optimization calculations were performed at the HF/6-31G level of
theory in the gas phase. The minimum geometries were fully optimized
at the B3LYP/6-311G (d) level of theory in the gas phase to yield more
accurate conformers. The ECD spectra were simulated at the B3LYP/6-
To the best of our knowledge, the separation and determina-
tion of concentration of FLU enantiomers in mariculture pond
water has not been carried out. Moreover, the degradation be-
havior of FLU enantiomers in mariculture pond water has not
been evaluated. In this work, a chiral high-performance liquid
chromatography (HPLC) method was developed and validated
for the determination of the concentrations of FLU enantio-
mers in mariculture pond water. The degradation behavior of
FLU enantiomers in mariculture pond water was investigated
*Correspondence to: Xiao-kun OuYang or Yang-Guang Wang, School of Food
and Pharmacy, Zhejiang Ocean University, Zhoushan 316022, P.R. China. E-
mail: xkouyang@zjou.edu.cn
Received for publication 26 April 2016; Accepted 24 June 2016
DOI: 10.1002/chir.22625
Published online in Wiley Online Library
(wileyonlinelibrary.com).
© 2016 Wiley Periodicals, Inc.

WANG ET AL.
under an average illumination intensity of 4.4 × 104 lx. Three analysis of
each sample were removed following treatments for different time inter-
vals and were immediately subjected to extraction.
Incubation of FLU Under Sterile Conditions
In order to determine whether the degradation of FLU was a result of
microbe-mediated transformations, batch experiments were conducted
in sterile mariculture pond water. rac-FLU (S:R=1:1, spike level of 10
mg/L) was added to sterile mariculture pond water. The spiked samples
were then transferred to 100-mL beakers, and the beakers were covered
with preservative films to prevent the evaporation of water. Finally, the
mariculture pond water samples were incubated at 34.5°C under an aver-
age illumination intensity of 4.4 × 104 lx in the solar simulator.
Fig. 1. Chemical structures of FLU enantiomers.
311G (d, p) level of theory in methanol.11,20 The calculated ECD curves
were generated using SpecDis with a σ of 0.2 eV.
Sample Preparation
The mariculture pond water samples were obtained from a mariculture
pond in ZhouShan City (Zhejiang, China) and were subjected to vacuum
filtration to remove suspended particles (e.g., dead creatures, sand, and
aerial dust). Further, the mariculture pond water was filtered through a
0.22 μm microporous membrane to yield sterile samples. The samples
were then transferred to 1-L glass bottles and stored in the dark at 4°C
until analysis, which occurred within 2 weeks. The FLU level in the mari-
culture pond water samples was less than the limit of detection (LOD) of
the HPLC method. Further details regarding the mariculture pond water
samples and specific physicochemical characteristics such as pH, total
bacteria count, total organic carbon, and salinity are listed in Table 1.
All the mariculture pond water samples were placed in a solar simulator
Suntest CPS+ (Heraeus, Germany) for the photolysis experiments.
Incubation of FLU Under Dark Conditions
In order to investigate the effect of light on the degradation of FLU en-
antiomers, mariculture pond water samples were spiked with rac-FLU (S:
R=1:1, spike level of 10 mg/L) and transferred to beakers. The beakers
were covered with aluminum films and then incubated in the solar simu-
lator at 34.5°C. At every sampling timepoint, the samples were analyzed
in triplicate.
Chromatographic Conditions
For method development and validation, HPLC was carried out with an
Agilent 1200 HPLC system (Agilent, palo Alto, CA) consisting of a quater-
nary pump (G1311A), degasser unit (G1322A), variable wavelength de-
tector (G1314B), and manual injector (G1328B). Chiralpak OD-H (4.6
mm × 250 mm i.d., 5 μm) was employed as the column. The mobile phase
consisted of n-hexane-ethanol (20:80, v/v) at a flow rate of 0.6 mL/min.
Acetic acid (0.2%) and ammonium acetate (5 mmol/L, 0.2% (v/v)) were
used as additives in the mobile phase. The UV detector was set at 254
nm, and the injection volume was 20 μL. The HPLC system was con-
trolled and data analyzed using a computer equipped with Chemstation
software (Rev.B.04.03, Agilent).
Extraction and Cleanup Procedure
The mariculture pond water samples (3 mL) were run through an Oasis
HLB cartridge (150 mg, 3 mL, Waters, Milford, MA) that was first condi-
tioned with 5 mL of methanol and deionized water. The flow rate was 2
mL/min. Thereafter, 5 mL of deionized water was used to remove the im-
purities in the samples and 5 mL of methanol was used to elute the
retained analyte. The eluates were collected in a glass test tube and evap-
orated to dryness under a gentle stream of nitrogen in a water bath at 30°
C. The obtained residues were then dissolved in 3 mL of ethanol. Prior to
analysis, all the samples were filtered through a 0.22 μm nylon syringe fil-
ter (Agilent Technologies, Germany) and a 20 μL aliquot of the resulting
solution was injected into the HPLC system.
Method Development and Validation
S-(ꢀ)-FLU and R-(+)- FLU were accurately weighed, transferred to a
volumetric flask, and dissolved in ethanol to prepare individual stock so-
lutions with concentrations of 20 mg/L. These solutions were thoroughly
mixed and stored at 4°C in tightly closed bottles until use. Working solu-
tions of S-(ꢀ)-FLU and R-(+)-FLU at concentrations of 0.05 mg/L, 0.25
mg/L, 1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, and 5 mg/L were diluted with
ethanol to prepare the stock solutions. Calibration curves were generated
by plotting the area under the peak in the HPLC chromatograms of the
enantiomers against the actual concentration of each enantiomer used.
The LOD and limit of quantitation (LOQ) of the mariculture pond water
samples were established at signal-to-noise ratios (S/Ns) of 3 and 10,
respectively.
Stock solution was diluted with blank mariculture pond water to give fi-
nal concentration of 0.1 mg/L, 2.5 mg/L, and 4.0 mg/L. These spiked mari-
culture pond water samples were considered the quality control (QC)
samples. For the method recovery studies, QC samples (3 mL) were ana-
lyzed using the developed HPLC method and the recovery values were
subsequently calculated. Accuracy was expressed as recovery, and preci-
sion was expressed as intra- and interday relative standard deviation
(RSD). Intraday RSD was determined by five replicate analyses of the
QC samples on 1 day. Interday RSD was determined by five replicate anal-
yses of the QC samples on 5 different days within a 14-day period.
Configurational Stability of Pure S-(ꢀ)-FLU and R-(+)-FLU
The configurational stability of each enantiomer was evaluated in tripli-
cate by analyzing mariculture pond water samples spiked with two con-
centration levels of pure S-(ꢀ)-FLU or R-(+)-FLU (0.5 mg/L and 5
mg/L). The spiked samples were incubated under natural, sterile, and
dark conditions. After a certain incubation period (1, 10, 20, or 30 h for
5 mg/L samples and 1, 5, 10, or 20 h for 0.5 mg/L samples), S-(ꢀ)-FLU
and R-(+)-FLU were extracted from the spiked samples and analyzed via
the aforementioned methods.
Incubation of FLU Under Natural Conditions
Batch incubation experiments were conducted with the mariculture
pond water samples (1.5 L) spiked with rac-FLU (S:R=1:1, spiking level
of 10 mg/L). Equal volumes of the spiked mariculture pond water were
then transferred to 15 100-mL beakers, which were covered with preser-
vative aluminum films to prevent evaporation of the water. Subsequently,
the mariculture pond water samples were placed in a solar simulator
Suntest CPS+ for the photolysis experiments and incubated at 34.5°C
TABLE 1. Physicochemical characteristics of the mariculture pond water samples
Characteristics
Values
CODa (mg/L)
5.0
TOCb (mg/L)
1.7
Salinity (‰)
APCc (cfu/mL)
6.8×104
Temperature (°C)
34.5
Illumination intensity (lx)
4.4×104
pH
8.1
34.0
aChemical oxygen demand.
bTotal organic carbon.
cAerobic plate count.
Chirality DOI 10.1002/chir

DEGRADATION BEHAVIOR OF FLUMEQUINE ENANTIOMERS
Fig. 2. Calculated (A) and experimental (B) ECD spectra of FLU enantiomers in methanol.
Kinetic Analysis and Calculations
RESULTS AND DISCUSSION
Absolute Configuration of FLU Enantiomers
For all the conditions, the data were assumed to follow a first-order ki-
netic model. The corresponding rate constants, k, for S-(ꢀ)-FLU or R-(+)-
FLU were calculated using eq. 1 by regression analysis. The half-life
(t1/2, h) was determined using eq. 2.
The absolute configuration of a chiral molecule can be de-
duced from its ECD spectrum.21 In this study, TDDFT20
was used to calculate the ECD spectra of FLU based on the
preferred conformation of FLU enantiomers (which was ob-
tained using Gaussian 09, Supporting Information 1). By com-
paring the experimental ECD spectra to the calculated
spectra, the absolute configurations of the FLU enantiomers
were determined. The overall ECD curves obtained by the
TDDFT calculations (Fig. 2A) and those obtained experimen-
tally (Fig. 2B) were similar. In general, individual enantio-
mers exhibited specific CD signals at given wavelengths.
The first eluted enantiomer was confirmed as the (ꢀ)-enantio-
mer, whereas the second enantiomer was the (+)-enantiomer,
as noted by the region ranging from 200 to 500 nm (Fig. 2B).
Through the combination of computed and experimental
lnðC=C0Þ ¼ ꢀkt
t1=2 ¼ 0:693=k
(1)
(2)
where C and C0 are the concentrations of S-(ꢀ)-FLU or R-(+)-FLU at
time t and time 0, respectively, and k is the degradation rate constant of
S-(ꢀ)-FLU or R-(+)-FLU.
The enantiomer concentration ratio (ER), calculated using eq. 3, was
used to assess the enantioselectivity in this study. Enantioselectivity
was confirmed by ER values not equal to 1.0.
ER ¼ concentration of ½Sꢁ=½Rꢁ
(3)
Fig. 3. (A) Representative HPLC chromatogram of S-(ꢀ)-FLU in a standard solution. (B) Representative HPLC chromatogram of R-(+)-FLU in a standard solu-
tion. (C) Chromatogram of blank mariculture pond water. (D) Chromatogram of rac-FLU in mariculture pond water. Peak assignment: 1: S-(ꢀ)-FLU; 2: R-(+)-FLU.
Chirality DOI 10.1002/chir

WANG ET AL.
TABLE 2. Effect of drip washing and elution volume on the
recovery of FLU enantiomers
mg/L, 0.25 mg/L, 1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, and 5
mg/L of each enantiomer). The calibration curves were gen-
erated by plotting the peak area (y) against the amount of
each analyte injected (x, mg/L). As shown in Table 3, the cal-
ibration curves were linear over the investigated concentra-
tion ranges, with correlation coefficients (R2) greater than
0.999. The LODs were 0.016 mg/L and 0.016 mg/L for
S-(ꢀ)-FLU and R-(+)-FLU, respectively, in the mariculture
pond water samples, whereas the LOQs were 0.05 mg/L for
both the enantiomers. The recovery of the method was evalu-
ated by performing five replicates of three spiked QC samples
(0.1 mg/L, 2.5 mg/L, and 4 mg/L). As shown in Table 4,
good recovery values between 88.3% and 95.2% were ob-
tained. These values not only confirmed the accuracy of the
method, but also highlighted the integrity of the SPE proce-
dure. Intraday precision was evaluated by analyzing repli-
cates of three spiked QC samples (0.1 mg/L, 2.5 mg/L, and
4 mg/L). The intraday precision (RSD) values were deter-
mined to be between 4.14% and 7.78% (shown in Table 4).
Interday precision (RSD) was evaluated by analyzing five rep-
licates of three spiked QC samples (0.1 mg/L, 2.5 mg/L, and
4 mg/L) on 5 different days within a 2-week period and the
values were calculated to be between 5.31% and 8.24% (shown
in Table 4). These small RSD values indicated good reproduc-
ibility and high accuracy.
Recovery (%)a
Amount
(mL)
Procedure
S-(ꢀ)-FLU
R-(+)-FLU
Drip washing
3
88.3 5.32
94.3 4.78
91.5 2.44
84.6 3.41
93.3 5.73
92.4 5.46
91.5 6.38
96.3 7.23
95.2 4.25
85.8 4.63
94.6 2.50
92.6 4.75
5
7
3
5
7
Elution
aExpressed as mean SD
ECD spectra, peak 1 and peak 2 in Figure 3 were assigned to
S-(ꢀ)-FLU and R-(+)-FLU, respectively.
Solid Phase Extraction (SPE) Optimization
SPE was applied for the extraction and cleanup of FLU in
the mariculture pond water samples. The effects of the drip
washing volume and elution volume were investigated and
the recovery values were determined. The mariculture pond
water samples (3 mL) were run through an Oasis HLB car-
tridge (150 mg, 3 mL, Waters), using an adequate amount
of deionized water to wash away the impurities in the sam-
ples. However, the use of excess deionized water reduced
the recovery of FLU enantiomers. Therefore, the recovery
of FLU enantiomers as a function of drip washing volume
was investigated. As shown in Table 2, when the drip washing
volume was 3 mL, the recovery of FLU enantiomers was rela-
tively high, but numerous impurities were detected in the
HPLC results. When the drip washing volume was 7 mL,
fewer impurities were observed, but a lower recovery was ob-
tained. Finally, 5 mL of deionized water was chosen as the op-
timal drip washing volume. The elution volume was also an
important factor in SPE. Table 2 shows the relationship
between elution volume and recovery of the FLU enantio-
mers. When the elution volume was 3 mL, the recoveries of
S-(ꢀ)-FLU and R-(+)-FLU were 84.6% and 85.5%, respectively.
However, the use of 5 and 7 mL elution volumes yielded bet-
ter results as the recoveries of S-(ꢀ)-FLU and R-(+)-FLU were
greater than 92.4%. Although large elution volumes could be
used to completely elute the target compounds, the possibil-
ity of losing the targets due to the flow of nitrogen also in-
creased. Therefore, an elution volume of 5 mL was selected.
Configurational Stability of FLU Enantiomers
The absolute configurations of some chiral drugs, such as
phenthoate and triadimefon, could be inverted.13 However,
in this work no measurable levels of the other enantiomer
were detected following the pure enantiomer incubation
experiments.
The results showed that the configurations of the enantio-
mers were not inverted.
Degradation of FLU in Mariculture Pond Water
Plots of the initial concentration of FLU enantiomers versus
incubation time (t) under natural conditions are shown in
Figure 4A. As shown in Figure 4A, the degradation of FLU en-
antiomers continuously decreased with time, i.e., the plot of
ln(C) versus t was linear (Fig. 4B). The results suggest that
the degradation of FLU enantiomers followed first-order ki-
netics under natural conditions, with correlation coefficients
ranging from 0.9589 to 0.9626 (Table 5). The t1/2 value for
S-(ꢀ)-FLU was 5.05 h, whereas the t1/2 value for R-(+)-FLU
was 5.21 h. In other words, the degradation of S-(ꢀ)-FLU oc-
curred slightly faster than that of R-(+)-FLU. The ERs
resulting from the degradation of rac-FLU under natural con-
ditions are plotted in Figure 4C. During the early stages of the
incubation experiments, the ERs were constant at a value of
Validation of the Chiral HPLC Method
The chiral HPLC method was validated and the linearity,
LODs, and LOQs were determined via serial dilution of the
standard solutions containing the FLU enantiomers (0.05
TABLE 3. Linear range, correlation coefficient, limit of detection (LOD), and limit of quantitation (LOQ) for FLU enantiomers
Regression
equationa
Linear range
(mg/L)
Correlation coefficient
Limit of detectionb
(mg/L)
Limit of quantitationc
(mg/L)
Analyte
(R2)
S-(ꢀ)-FLU
R-(+)-FLU
y=115.35 x-10.98
y=119.43 x-14.493
0.025-5.00
0.025-5.00
0.9995
0.9996
0.016
0.016
0.05
0.05
ay: peak area, x: the concentration of analyte (mg/L).
bLimit of detection: S/N>3.
cLimit of quantitation: S/N>10.
Chirality DOI 10.1002/chir

DEGRADATION BEHAVIOR OF FLUMEQUINE ENANTIOMERS
TABLE 4. Recoveries and precision for the FLU enantiomers.
about 1.0. After incubation for 20 and 10 h, the ER values ex-
hibited a slight decrease over time from the initial value of 1.0
to 0.88 (concentration ratio of S/R), as shown in Figure 4C.
The ER values suggested that S-(ꢀ)-FLU degraded more rap-
idly than R-(+)-FLU.
Plots of the initial concentrations of FLU enantiomers ver-
sus t under sterile conditions are shown in Figure 4D. As
shown in Figure 4D, the degradation of FLU enantiomers de-
creased continually with time. The plot of ln(C) versus t was
linear (Fig. 4E), suggesting that the degradation of the enan-
tiomers followed first-order kinetics under sterile conditions
with correlation coefficients ranging from 0.9811 to 0.9831
(Table 5). The t1/2 value of S-(ꢀ)-FLU was 6.54 h, whereas
that of R-(+)-FLU was 6.72 h for the pond water samples.
Recovery (n = 5)
Mean(%) SDa
Precision (RSD %)
Spiked level
(mg/L)
Analyte
Intradayb Interdayc
S-(ꢀ)-FLU
0.1
2.5
4.0
0.1
2.5
4.0
88.4 6.25
92.9 8.48
91.4 7.28
92.2 6.52
95.2 6.18
89.7 5.12
5.40
5.71
4.74
7.78
4.14
4.57
6.48
8.24
7.69
6.76
6.17
5.31
R-(+)-FLU
aDetermination in 1 day.
bn = 5.
cn = 5 replicates×5 days within 14 days.
Fig. 4. Degradation of FLU enantiomers (10 mg/L of rac-FLU) in mariculture pond water under natural (A), sterile (D), and dark (G) conditions. Plot of ln(C)
versus incubation time t under natural (B) and sterile (E) conditions (10 mg/L for rac-FLU). Plot of ER (where ER=[S]/[R]) versus incubation time t under natural
(C) and sterile (F) conditions (10 mg/L for rac-FLU).
Chirality DOI 10.1002/chir

WANG ET AL.
TABLE 5. Degradation rate constant (k), half-life (t1/2), and
pond water had some effect on the degradation behavior.
These results are expected to contribute to the understanding
of the fate of FLU enantiomers in mariculture pond water and
aid in evaluating the potential risks of FLU to mariculture
environments.
correlation coefficient (R2) values for the degradation of rac-
FLU under natural and sterile conditions
Conditions
Natural
Enantiomers
k (h-1)
t1/2 (h)
R2
S-(ꢀ)-FLU
R-(+)-FLU
S-(ꢀ)-FLU
R-(+)-FLU
0.14
0.13
0.11
0.10
5.05
5.21
6.54
6.72
0.9626
0.9589
0.9811
0.9831
ACKNOWLEDGMENTS
Sterile
This work was financially supported by the Zhejiang Pro-
vincial Natural Science Foundation of China (LY14C200003),
National Natural Science Foundation of China (21276240),
Funds of Science and Technology Department of Zhejiang
province (2014C32080), Project of Zhejiang Education De-
partment (PD2013224).
Comparison of these values with the t1/2 values of the FLU en-
antiomers for pond water samples under natural conditions
revealed that the FLU enantiomers degraded more slowly un-
der sterile conditions. This suggested that the microbial com-
munity in the mariculture pond water was able to accelerate
the degradation of the FLU enantiomers. The ER values ob-
tained by the degradation of rac-FLU under sterile conditions
are plotted in Figure 4F. While the ER values were stable at
about 1.0 during the initial stages of the experiments, they de-
creased continuously over time. For example, after incubation
for 16 and 8 h, the ER values were reduced to 0.90 and 0.84
(concentration ratio of R/S), respectively, suggesting that
S-(ꢀ)-FLU degraded more rapidly than R-(+)-FLU, and a
slight increase in one FLU enantiomer in the mariculture
pond water samples was observed, as indicated by the ER
values. The slight changes in the t1/2 and ER values were at-
tributed to inevitable experimental error and we concluded
that the degradation behavior of FLU enantiomers under ster-
ile conditions was not enantioselective.
Plots of the initial concentrations of FLU enantiomers ver-
sus t under dark conditions are shown in Figure 4G. The con-
centration of FLU enantiomers did not change with time,
suggesting that S-(ꢀ)-FLU and R-(+)-FLU did not degrade un-
der dark conditions. The results suggest that light had a signif-
icant effect on the degradation rate of the FLU enantiomers.
Based on the results of the experiments conducted under
different conditions, it was concluded that the degradation
rates of the FLU enantiomers were greatest under natural con-
ditions, followed by sterile conditions. The enantiomers did
not degrade in the dark, indicating that light and microbial
community in the mariculture pond water contributed to the
degradation of FLU enantiomers. The degradation of the
FLU enantiomers varied depending on the conditions (natural
vs. sterile), suggesting that microorganisms contributed to
the degradation of FLU enantiomers, but did not contribute
to the enantioselective degradation of FLU enantiomers in
the mariculture pond water. However, rac-FLU did not de-
grade under dark conditions, and enantioselective degrada-
tion was not observed in the dark.
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In conclusion, a chiral HPLC method was established for the
simultaneous detection of S-(ꢀ)-FLU and R-(+)-FLU enantio-
mers in mariculture pond water. The extraction, purification,
and chromatographic parameters for sample pretreatment
were experimentally optimized. Degradation of the FLU enan-
tiomers in mariculture pond water samples under different
conditions was investigated. The degradation of rac-FLU in
mariculture pond water under natural, sterile, and dark condi-
tions showed no enantioselectivity. Photodegradation ap-
peared to be the primary mode of degradation of FLU
enantiomers and the microbial community in the mariculture
Chirality DOI 10.1002/chir
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