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LI ET AL.
and LOQ (limit of quantification) of the enantioselective analysis were
0.007 mg/g and 0.02 mg/g, respectively.
Nonenantioselective and Enantioselective Analysis
The nonenantioselective and enantioselective analysis procedure was
reported in our previous papers.17,21 A liquid chromatography apparatus
(Shimadzu, Kyoto, Japan) consisted of a LC-20AB pump, a LC-20A UV de-
tector set at 225 nm and an LC-solution chemical analytical work station.
The injection volume was 20 mL. Chromatographic separation was
conducted at 25ꢀC and the flow rate was maintained at 1.0 mL/min.
An achiral silica-gel column (Dalian Elite Co. Ltd., China, 250ꢃ 4.6 mm
i.d., 5 mm particle size) with a guard cartridge of the same phase (30 ꢃ 4.6 mm
i.d.) was used for the quantification of triadimenol-A and triadimenol-B in
soil. The mobile phase was an isocratic mixture of 100:2.5 n-hexane/
isopropanol (v/v) and the HPLC system could resolve triadimenol-B and
triadimenol-A at 17.8 and 22.0 min, respectively. For the enantioselective
analysis, another clean-up procedure was necessary. The remainder sam-
ples after quantification were concentrated to about 0.08 mL and injected
into the silica-gel column via four consecutive injections. Mobile phase frac-
tions containing triadimenol-B and triadimenol-A were collected manually
by observing their UV signals. After being gently blown to dryness and
dissolved in 0.1 mL n-hexane/isopropanol (100:2.7, v/v), the samples were
subjected to enantioselective HPLC. In this procedure, a Chiralcel OD-H
column (Daicel Chemical Industries Ltd., City?, Japan, 250ꢃ 4.6 mm i.d., 5
mm particle size) and a Chiralcel OJ-H column (Daicel Chemical Industries
Ltd., 250ꢃ 4.6 mm i.d., 5 mm particle size), each protected with a guard col-
umn of the same phase, were used. The enantiomer separation conditions
were as follows. Triadimenol-A: Chiralcel OD-H column, 100:2.7 n-hexane/
isopropanol (v/v) for triadimenol-A, 1R,2S-triadimenol and 1S,2R-
triadimenol were eluted at 16.4 and 20.5 min, respectively; Triadimenol-B:
Chiralcel OJ-H column, 100:2.7 n-hexane/isopropanol (v/v) for triadimenol-
B, 1R,2R-triadimenol and 1S,2S-triadimenol were eluted at 21.8 and 24.3
min, respectively. No interconversion between stereoisomers was detected
during the extraction and HPLC separation processes.
Fig. 1. Chemical structure of triadimenol.
Institute (Shanghai, China). Triadimenol-A (>99%) was purchased from
Dr. Ehrenstorfer (Augsburg, Germany) and triadimenol-B (>99%) was from
Jiangsu Sword Agrochemicals Co. Ltd. (City, China). Enantiopure 1S,2R-
triadimenol and 1R,2S-triadimenol were prepared in this study via enantio-
meric resolution of racemic triadimenol-A by chiral HPLC (see below). In
this step, triadimenol-A of known quantity was injected into the chiral HPLC
system and the mobile phase fractions corresponding to 1S,2R-triadimenol
and 1R,2S-triadimenol were collected manually by observing their UV
signals. The collected samples were then gently concentrated to dryness un-
der
a vacuum evaporator and used as enantiomer standards. The
enantiopurity of the prepared stereoisomers, checked with chiral HPLC
using the same column, was >99%.
Florisil (60–100 mesh) was activated at 630ꢀC for 4 h before use, and was
obtained from Sigma-Aldrich (St Louis, MO, USA). Anhydrous sodium sul-
fate was analytical grade, heated at 450ꢀC for 4 h before use. n-Hexane,
isopropanol, and ethyl acetate were all of analytical grade, redistilled and fil-
tered through a 0.45 mm nylon filter membrane before use.
An agricultural yellow soil from Baoding city in North China and a
garden red soil from Wuhan city in South China were used in this study.
The soil samples were air-dried and stocked in the dark and sifted out via
a griddle of 20-mesh before use. The two soils were slightly alkaline and
slightly acidic with pH values of 7.86 and 6.25, respectively. Other soil
properties were as follows: Baoding soil (SB): organic matter 1.82%, sand
50.59%, silt 31.70%, and clay 17.71%; Wuhan soil (SW): organic matter
4.22%, sand 20.57%, silt 16.57%, and clay 62.86%. Blank determination of
the soils prior to fortification revealed no triadimenol (detection limit,
0.02 mg/g) present. When sterilized experiments were performed, the
soils, water, and glassware used were autoclaved at 120ꢀC twice (4 h
per time) on consecutive days.
The enantiomeric fraction (EF) was used as a measure of the
enantioselectivity and EF is defined by equation 1:
EF ¼ A1=ðA1 þ A2Þ
(1)
where A1 and A2 are peak areas of the first eluted enantiomer and late
eluted enantiomer, respectively, assuming the same response factors
for enantiomers originating from the same compound. The EF values de-
fined the range from 0 to 1, with EF = 0.5 representing the racemic mix-
ture. Based on the concentrations of triadimenol-A and triadimenol-B,
and corresponding EF values, the concentrations of individual stereoiso-
mers in soil were then calculated.
Soil Incubation Experiments
Separate experiments were carried out with technical triadimenol and
selective single stereoisomers in the two soils. In each experiment, a por-
tion of 10 g dry soil was transferred into a 250 mL conical flask and spiked
with 0.3 mL acetone containing 550 mg technical triadimenol (experi-
ments SB1 and SW1 for the Baoding soil and Wuhan soil, respectively),
275 mg triadimenol-B (experiments SB2 and SW2, respectively), 137 mg
1S,2R-triadimenol (experiments SB3 and SW3, respectively) or 137 mg
1R,2S-triadimenol (experiments SB4 and SW4, respectively), and stirred
thoroughly for 10 min to allow acetone evaporated. 100 g more soil was
added and mixed thoroughly, yielding a final fortification level of 5 mg/g
for technical triadimenol, 2.5 mg/g for triadimenol-B, or 1.25 mg/g for
1S,2R-triadimenol and 1R,2S-triadimenol, respectively. Then 28 mL distilled
water was added to give a ~20% final moisture content. After carefully
mixing, the flasks were sealed with cotton-wool plugs and incubated at
25 ꢁ 1ꢀC in the dark. To verify that the degradation was microbial, sterilized
control experiments (experiments SB1’–SB4’ and SW1’–SW4’) were also
performed as compared to corresponding nonsterilized experiments.
Distilled water was added at three or four day intervals to maintain the initial
moisture. Replicate soil samples equivalent to 8 g dry soil weight were
removed for analysis at 0.5 h after fortification (0 d), followed at different
time intervals, and frozen at ꢂ20ꢀC until they could be analyzed.
RESULTS AND DISCUSSION
Stereoselective Degradation and Microbial Epimerization of
Technical Triadimenol in Soil
In Figure 2a and 2b, we plotted the data for triadimenol-A and
triadimenol-B concentrations as a function of time from experi-
ments SB1 and SW1 with technical triadimenol. The initially
spiked concentrations for triadimenol-A and triadimenol-B were
3.44 mg/g and 0.60 mg/g or 3.42 mg/g and 0.58 mg/g for Bao-
ding soil or Wuhan soil, respectively. After 100 days incubation,
the triadimenol-A amount decreased continuously to a final 0.40
mg/g or 0.14 mg/g for the Baoding soil or Wuhan soil, respec-
tively. However, as for triadimenol-B, it is interesting that its
concentration increased in the former part of the incubation
and then decreased in the later stages. The concentration of
triadimenol-B reached a maximum of 1.36 mg/g or 1.72 mg/g
and exceeded that of triadimenol-A at 70 d or 50 d for Baoding
soil or Wuhan soil, respectively. The results clearly show
significant epimerization from triadimenol-A to triadimenol-B
occurred during the incubation process. To illustrate the
epimerization more clearly, the chromatograms in Figure 3
Extraction of Triadimenol from Soil
The extraction of triadimenol from soil was carried out by the matrix
solid-phase dispersion (MSPD) extraction method as reported in our pre-
vious paper.17 When fortified at 1.0 and 0.1 mg/g, average recoveries of
triadimenol (n = 3) from the MSPD extraction process were >75% with
relative standard deviations (RSDs) <10%. The LOD (limit of detection)
Chirality DOI 10.1002/chir
Li, Zhaoyang
