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SUPLEMMENTARY MATERIAL

In vitro enantioselective study of the toxicokinetic effects of chiral fungicide

tebuconazole in human liver microsomes

Maísa Daniela Habenschus1, Viviani Nardini1, Luís Gustavo Dias1, Bruno Alves Rocha2,

Fernando Barbosa Jr2, Anderson Rodrigo Moraes de Oliveira1*

1 Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão

Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto - SP, Brazil

2 Laboratório de Toxicologia e Essencialidade de Metais, Faculdade de Ciências

Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14049-903 Ribeirão

Preto, SP, Brazil

*Correspondence: Prof. Dr. Anderson Rodrigo Moraes de Oliveira. Departamento de

Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto - USP - Av. dos

Bandeirantes, 3900, Ribeirão Preto, São Paulo, 14040-901, Brazil

E-mail: [email protected]

Phone: +55-16-3315-3799

Fax: +55-16-3315-4838

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Chemical and Reagents

Chemical inhibitors used during CYP450 reaction phenotyping studies were

purchased from Sigma-Aldrich (St. Louis, MO, USA), and standard inhibitor stock

solutions were prepared in acetonitrile: diethyldithiocarbamate for CYP2E1 (≥ 99%,

8000 μmol L-1), ketoconazole for CYP3A4/5 (≥ 98%, 40 μmol L-1), ticlopidine for

CYP2B6 and CYP2C19 (≥ 99%, 240 and 800 μmol L-1, respectively), quinidine for

CYP2D6 (≥ 98%, 80 μmol L-1), α-naphtoflavone for CYP1A2 (≥ 98%, 80 μmol L-1),

montelukast for CYP2C8 (≥ 98%, 80 μmol L-1), and sulfaphenazole for CYP2C9 (≥

98%, 800 μmol L-1). All solutions were stored in amber tubes at −20 °C.

Reagents used to generate NADPH, β-nicotinamide adenine dinucleotide

phosphate hydrate (NADP+), glucose-6-phosphate dehydrogenase, and glucose-6

phosphate sodium salt, were acquired from Sigma Aldrich (St. Louis, MO, USA). These

reagents were prepared in tris-KCl buffer (tris(hydroxymethyl)aminomethane 0.05 mol

L-1 and KCl 0.15 mol L-1, pH 7.4) and stored at −20 °C at the following concentrations:

NADP+ at 2.5 mmol L-1, glucose-6-phosphate dehydrogenase at 8.0 units mL-1, and

glucose-6-phosphate at 50 mmol L-1. Sodium phosphate monobasic, sodium phosphate

dibasic, and hydrochloric acid were purchased from Synth (Diadema, SP, Brazil).

Tris(hydroxymethyl)aminomethane was acquired from J. T. Baker (Phillisburg, NJ,

USA), and potassium chloride was purchased from Mallinckrodt Chemicals (Xalostoc,

Mexico). Ultrapure water was obtained from a Direct-Q3 system (Millipore, Billerica,

MA, USA). HPLC grade solvents, methanol, acetonitrile, and ethyl acetate were

acquired from Panreac (Barcelona, Spain), whereas isopropanol and hexane were

obtained from Honeywell Riedel-de Häenm (Seelze, Germany).

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Tebuconazole and 1-hydroxytebuconazole enantioselective method development

The analytical method was developed to determine in vitro TEB metabolism

enantioselectively. To achieve a satisfactory resolution (Rs) between TEB and TEBOH

enantiomers (Rs ≥ 1.5), a screening analytical strategy based on Perrin et al., 2002 and

Matthijs et al., 2006 was adopted. Firstly, the Shimadzu HPLC- DAD system described

in section 2.2 was employed to evaluate the separation conditions with the diode array

detector operating at 220 nm and column temperature kept at 20 ± 2ºC. Chiral columns

with different lengths, chiral selectors and particle sizes were evaluated: Chiralpak AD-

H® (150 x 4.6 mm, 5 μm), Chiralpak AS® (250 x 4.6 mm, 10 μm), Chiralcel OD-H®

(150 x 4.6 mm, 5 μm), Chiralcel OJ® (250 x 4.6 mm, 10 μm), Lux Cellulose-1® (150 x

4.6 mm, 5 μm), Lux Cellulose-2® (150 x 4.6 mm, 5 μm), Lux Amylose-2® (150 x 4.6

mm, 5 μm) and Chirobiotic T® (150 x 4.6 mm, 5 μm), and different mobile phase

compositions and flow rates were tested respecting the specifications and maximum

pressure supported by each column.

The elution modes chosen to be evaluated were polar organic (PO-HPLC) and

reversed phased (RP-HPLC) elution modes. Both PO-HPLC and RP-HPLC offer

advantages compared to the normal phase, which is the most common elution mode

used for chiral separation, such as use of less toxic and less expensive solvents, better

polar analyte solubility in the mobile phase, and easier liquid chromatography system

coupling with mass spectrometry (Matthijs et al., 2006) (Perrin et al., 2002).

LC-MS/MS enables highly selective and sensitive methods to be developed, which is

very important for in vitro metabolism studies, during which low quantities of a given

metabolite may arise (Jing et al., 2013).

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In PO-HPLC mode, the mobile phases evaluated were methanol, ethanol,

isopropanol, acetonitrile and mixtures of these solvents. In RP-HPLC mode, mixtures of

methanol or acetonitrile with water were evaluated (Fig. S3B).

To optimize the process, some screening criteria were adopted. Initially, only the

rac-TEB was injected (20 μL at 100 μg mL-1) at the equipment to evaluate enantiomers

resolution. If the enantiomers of TEB were separated using the specific condition, then

rac-TEBOH was injected (20 μL at 50 μg mL-1) and its enantiomers separation

evaluated. Besides, if there were no indications of resolution of the enantiomers or the

compounds did not elute within 15 minutes, analysis was stopped and a new condition

was tested. Table S1 and Table S2 resume the analytical conditions evaluated during

the screening procedure for PO-HPLC and RP-HPLC, respectively.

After the screening procedure with HPLC-DAD, the most promising separation

conditions for chiral separation of TEB and TEBOH enantiomers in PO-HPLC and RP-

HPLC were obtained using Chiralcel OJ®, a column with a stationary phase based on

cellulose tris-(4-methyl benzoate). Elucidating chiral recognition models involved in

enantiomeric separation is a difficult task, but this finding may have been due to

hydrogen bonds formed between hydroxyl groups present in the TEB and TEBOH

chemical structures and the carbonyl group present in the chiral stationary phase (CSP)

chemical structure. The phenyl moiety may also have established π- π interactions with

the CSP aromatic ring (Zhou et al., 2009).

For PO-HPLC mode, the most promising separation condition was obtained with

100% methanol as the mobile phase at a flow rate of 0.5 mL min-1 and column

temperature of 20 ± 2 ºC (Fig. S4). For RP-HPLC mode, the most promising separation

conditions were accomplished with methanol: water (90:10, v/v) and acetonitrile: water

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(60:40, v/v) as mobile phases at flow rates of 0.5 mL min-1 and 0.7 mL min-1,

respectively, and column temperature of 20 ± 2 ºC (Fig. S5 and Fig. S6).

However, sensibility and selectivity problems were observed after the initial

metabolism pilot studies using the HPLC-DAD enantioselective methods previously

obtained. So, it was necessary to change the analytical technique to LC-MS/MS. After

the methods were transposed to the LC-MS/MS equipment and freshly prepared

metabolic samples were analyzed, two interferent peaks, with the same fragmentation

profile of TEBOH, were only resolved from TEBOH enantiomers with RP-HPLC

mode. Thus, analyte separation by LC-MS/MS was adjusted by reoptimizing the mobile

phase composition (methanol: water mixtures with and without formic acid, and

acetonitrile: water mixtures with and without formic acid), flow rate (0.1–0.6 mL min-1),

and column temperature (20–32 ºC).

Finally, chromatographic separation was carried out on a Chiralcel OJ® column

(250 x 4.6 mm, 10 μm) using methanol: water (90:10, v/v) as the mobile phase at a flow

rate of 0.6 mL min-1 and column temperature of 25 °C. The injection volume was 10 µL,

and the sample tray temperature was kept at 10 ºC. Capillary voltage was set at + 5000

V and capillary temperature was set at 320 °C. Nitrogen was used as the sheath gas and

auxiliary gas at flow rates of 10 and 5 (arbitrary units), respectively, and the vaporizer

temperature was set at 290 °C. Argon was used as a collision-induced dissociation gas

at 2.1 mTorr. Multiple reaction monitoring transitions were selected, and two reactions

were used for quantification (Q) and confirmation (C), with their respective collision

energies (CE) at m/z 308 → 70Q and 308 → 125C (CE 21 and 33 V) for TEB

enantiomers, m/z 324 → 70Q and 324 → 125C (CE 21 and 40 V) for TEBOH

enantiomers, and m/z 304 → 217Q and 304 → 202C (CE 23 and 34 V) for IS. Xcalibur

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software version 2.0 (Thermo Fisher Scientific) was used to control instruments and to

process data.

Method Validation

The enantioselective LC-MS/MS method was validated according to the EMA

guideline on Bioanalytical Methods validation (European Medicines Agency, 2012).

Linearity, selectivity, carryover, lower limit of quantification, inter- and intra-day

precision and accuracy, matrix effect, and stability were evaluated. Racemization of the

isolated TEB enantiomers submitted to incubation conditions was also assessed.

Linearity was assessed in triplicate by spiking a microsomal medium blank

(sodium phosphate buffer, Tris-KCl buffer, and HLM at protein concentration of 0.2 mg

L-1) with standard rac-TEB and rac-TEBOH stock solutions at final concentrations of

0.125, 0.250, 15.00, 25.00, 50.00, 75.00, and 85.00 μmol L-1 for each enantiomer of

TEB and 0.005, 0.015, 0.050, 0.250, 0.375, 0.750, and 0.900 μmol L-1 for each

enantiomer of TEBOH. Linear fits of the analytical curves were weighted by 1/x2 due to

the residual analysis heteroscedastic behavior, and linearity was expressed by the slope,

intercept, and correlation coefficient. ANOVA lack-of-fit tests using Minitab 16

Statistical software 7 (State College, PA, USA) were also performed to ensure linearity.

Selectivity was determined by analyzing blank microsomal medium samples for the

presence of co-eluted peaks at the same retention time of the analytes and IS. Carryover

was evaluated by analyzing blank sample chromatograms obtained right after the upper

limit of quantification was injected. Carryover was considered absent if interferences

that eluted at the same retention time as the analytes and IS represented ≤ 20% of the

lower limit of quantification area for TEB and TEBOH enantiomers and ≤ 5% of the IS

area. Intra-day (n = 5) and inter-day (n = 3) precision and accuracy were examined for

the lower limit of quantification and for the low-, medium-, and high-quality control

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samples. Results are expressed as the relative standard deviation (%RSD) for precision

and the relative error (%RE) for accuracy. %RSD and %RE were set within 15% and

±15% and 20% and ±20% for quality control samples and lower limit of quantification,

respectively. Matrix effect (n = 5) was evaluated by analyzing (i) blank samples spiked

with rac-TEB, rac-TEBOH, and IS after sample preparation procedures and (ii) rac-

TEB, rac-TEBOH, and IS solubilized in the mobile phase. Solutions were prepared at

the same concentrations as the low- and high-quality control samples. The normalized

matrix factor (NMF) was calculated by the ratio between TEB enantiomers and IS and

TEBOH enantiomers and IS peak areas, obtained from analyses of samples (i) and (ii)

(Eq. S1). In addition, the NMF %RSD should not be greater than 15%.

FMN=

Analyte peak area∈the blank sampleIS peak area∈theblank sample

Analyte peak area∈the mobile phaseIS peak area∈the mobile phase

(S1)

Analyte stability (n = 5) was evaluated for the low- and high-quality control

samples (i) under incubation conditions (30 min at 37 ºC) and (ii) in the LC-MS/MS

sample tray (48 h at 10 ºC). Samples were quantified by employing analytical curves

prepared on the same day, and they were considered stable if concentrations obtained

for each analyte were within ±15% of the nominal concentration. Finally, racemization

was assessed by incubating the isolated TEB enantiomers (n = 5) at two concentration

levels (2 and 50 µmol L-1) with HLM (0.2 mg mL-1) at 37 ºC for 20 min without

addition of NADPH regenerating system. Results were analyzed qualitatively by

observing the appearance of the other enantiomer in the chromatogram.

Analytical curves were linear over the concentration ranges of 0.125 to 85 µmol

L-1 for (+)- and (−)-TEB and of 0.005 to 0.900 µmol L-1 for (+)- and (−)-TEBOH, as

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confirmed by the ANOVA lack of fit test. Table S3 lists linear equations, correlation

coefficients, and ANOVA lack of fit parameters.

The method proved to be selective—no interference peaks emerged in the

analyte retention times (Fig. 1) after blank HLM samples were analyzed. Carryover was

evaluated by analyzing blank HLM samples right after the upper limit of quantification

was injected, and no carryover effect was observed (data not shown). The matrix effect

was investigated, and normalized matrix factor (NMF) values were calculated. Results

showed that %RSD was not higher than 9% (Table S4).

The method limit of quantification was 0.125 µmol L-1 for (+)- and (−)-TEB and

0.005 µmol L-1 for (+)- and (−)- TEBOH with %RSD lower than 11% and %RE lower

than 5%. Intra- and inter-day precision and accuracy were assessed, and the results are

in agreement with the EMA guideline requirements, as shown in Table S5. Stability

results showed that (+)-TEB, (−)-TEB, (+)-TEBOH, and (−)-TEBOH were stable when

they were kept in the equipment autosampler at 10 ºC for 48 h and under incubation

conditions (at 37 ºC for 30 min) (Table S6). Finally, no TEB enantiomer racemization

occurred under the incubation conditions (at 37 ºC for 20 min) (Fig. S7).

REFERENCES

European Medicines Agency, 2012. Guideline on bioanalytical method validation. EMA Guidel. 44, 1–23. https://doi.org/EMEA/CHMP/EWP/192217/2009

Jing, W.-H., Song, Y.-L., Yan, R., Wang, Y.-T., 2013. Identification of cytochrome P450 isoenzymes involved in metabolism of (+)-praeruptorin A, a calcium channel blocker, by human liver microsomes using ultra high-performance liquid chromatography coupled with tandem mass spectrometry. J. Pharm. Biomed. Anal. 77, 175–188. https://doi.org/10.1016/J.JPBA.2013.01.023

Matthijs, N., Maftouh, M., Heyden, Y. Vander, 2006. Screening approach for chiral separation of pharmaceuticals: IV. Polar organic solvent chromatography. J. Chromatogr. A 1111, 48–61. https://doi.org/10.1016/j.chroma.2006.01.106

Perrin, C., Matthijs, N., Mangelings, D., Granier-Loyaux, C., Maftouh, M., Massart, D.L., Vander Heyden, Y., 2002. Screening approach for chiral separation of pharmaceuticals: Part II. Reversed-phase liquid chromatography. J. Chromatogr. A

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966, 119–134. https://doi.org/10.1016/S0021-9673(02)00746-X

Zhou, Y., Li, L., Lin, K., Zhu, X., Liu, W., 2009. Enantiomer separation of triazole fungicides by high-performance liquid chromatography. Chirality 21, 421–427. https://doi.org/10.1002/chir.20607

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Table S1- Evaluated conditions during the analytical screening procedure by HPLC-DAD polar organic elution mode. The temperature of the columns was kept at 20±2ºC.

Chiral Column

Mobile PhaseComposition

Mobile Phase Proportion (% v/v)

Flow rate(mL min-1)

Resolution ofTEB

enantiomers

Resolution of TEBOH

enantiomersComments

Chiralpak AD-H®

Acetonitrile 100 0.3 0 - -

Acetonitrile: Methanol 50:50 0.3 0 - -

Acetonitrile: Ethanol 50:50 0.3 0.87 -It was not possible to increase the percentage of ethanol due to the

maximum column pressure allowed

Acetonitrile: Isopropanol

70:30 0.3 0 - -

50:50 0.2 1.06 -It was not possible to increase the

percentage of isopropanol due to the maximum column pressure allowed

Methanol 100 0.3 0 - -

Methanol: Ethanol 50:50 0.3 0 - -

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1920

Methanol: Isopropanol 50:50 0.3 0 - -

Ethanol 100 0.3 0 - -

Ethanol: Isopropanol 50:50 0.3 0 - -

Isopropanol 100 0.3 0 - -

Chiralpak AS®

Acetonitrile 100 0.3 0.37 - -

Acetonitrile: Methanol

85:15 0.3 0 - -

50:50 0.4 0 - -

Acetonitrile: Ethanol 85:15 0.3 0 - -

Acetonitrile: Isopropanol 85:15 0.3 0 - -

Methanol 100 0.4 0 - -

112122



Ethanol 100 0.4 0 - -


Isopropanol 100 0.3 - - It was not possible to evaluate due to column high pressure

Chiralcel OD-H®





Methanol 100 0.3 0 - -

122324



Ethanol 100 0.3 0 - -


Isopropanol 100 0.15 - - No peaks eluted until 15 minutes

Chiralcel OJ® Acetonitrile 100 0.5 >1.5 >1.5

E1 TEBOH and E1 TEB enantiomers and E2 TEBOH and E2 TEB

enantiomers coeluted

Acetonitrile: Methanol 85:15 0.5 >1.5 >1.5 E1 TEBOH and E1 TEB enantiomers coeluted

Acetonitrile: Ethanol 85:15 0.5 >1.5 >1.5 E1 TEBOH and E1 TEB, E2 TEBOH and E2 TEB enantiomers coeluted

132526


Methanol 100 0.5 >1.5 >1.5E1 TEBOH and E1 TEB enantiomers

coeluted (Fig. S4)- Promising separation condition

Methanol: Ethanol 50:50 0.5 >1.5 >1.5 E1 TEBOH and E1 TEB enantiomers coeluted

Methanol: Isopropanol 50:50 0.5 >1.5 >1.5 E1 TEBOH and E1 TEB enantiomers coeluted

Ethanol 100 0.5 >1.5 >1.5 E1 TEBOH and E1 TEB enantiomers coeluted

Ethanol: Isopropanol 50:50 0.5 >1.5 >1.5 E1 TEBOH and E1 TEB enantiomers coeluted

Isopropanol 100 0.3 >1.5 >1.5 E1 TEBOH, E1 TEB and E2 TEBOH enantiomers coeluted

Lux Cellulose-


142728

1®




Methanol 100 0.3 0 - -



Ethanol 100 0.3 0 - -



Lux Acetonitrile 100 0.5 >1.5 - E1 TEB eluted early and E2 TEB eluted

152930

Amylose-2®

after 15 minutes


Acetonitrile: Ethanol 85:15 0.5 >1.5 >1.5E1 TEBOH, E1 TEB and E2 TEBOH

enantiomers coeluted; E2 TEB showed very long retention time

Acetonitrile: Isopropanol

85:15 0.5 >1.5 >1.5 E1 TEBOH and E1 TEB enantiomers coeluted

70:30 0.5 >1.5 >1.5 E2 TEBOH and E1 TEB enantiomers coeluted

50:50 0.5 >1.5 1.27 -

Methanol 100 0.5 0 - -



163132

Ethanol 50:50 0.5 0 - -

Ethanol: Isopropanol 50:50 0.3 >1.5 0 -


Table S2- Evaluated conditions during the analytical screening procedure by HPLC-DAD reversed phase elution mode. The temperature of the columns was kept at 20±2ºC.

Chiral Column

Mobile PhaseComposition

Mobile Phase Proportion (% v/v)

Flow rate(mL min-1)

Resolution ofTEB

enantiomers

Resolution of TEBOH

enantiomersComments

Chiralpak AD-H®

Methanol: Water 90:10 0.6 >1.5 - Poor peak shapes and symmetry

Acetonitrile: Water 85:15 0.6 0.99 - -

75:25 0.6 1.14 - Poor peak shapes and symmetry

Chiralpak AS®

Methanol: Water

85:15 0.8 0.64 - -

80:20 0.8 0.89 - -

70:30 0.8 1.30 - Peaks eluted after 15 minutes

Acetonitrile: Water85:15 0.8 0.17 - -

80:20 0.8 0.22 - -

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70:30 0.8 0.34 - -

50:50 0.8 0.84 - Peaks eluted after 15 minutes

Chiralcel OD-H®

Methanol: Water85:15 0.6 0 - -

70:30 0.6 0 - -

Acetonitrile: Water85:15 0.6 0 - -

70:30 0.6 0 - -

Chiralcel OJ®

Methanol: Water90:10 0.5 >1.5 >1.5 Promising separation condition (Fig. S5)

85:15 0.5 >1.5 >1.5 E1 TEB and E2 TEBOH coeluted

Acetonitrile: Water



70:30 0.7 >1.5 >1.5 Promising separation condition (Fig. S6)

Lux Cellulose-1®

Methanol: Water 85:15 0.6 >1.5 - Poor peak shapes and symmetry

Acetonitrile: Water85:15 0.6 0 - -

70:30 0.6 0 - -

Lux Cellulose-2® Methanol: Water

85:15 0.6 0 - -

75:25 0.6 0 - -

183536

Acetonitrile: Water

85:15 0.6 0 - -

70:30 0.6 0.21 - -

60:40 0.6 0.42 - -

50:50 0.6 0.28 - -

Lux Amylose-2®

Methanol: Water85:15 0.7 0 - -

60:40 0.7 0 - Peaks eluted after 15 minutes

Acetonitrile: Water 75:25 0.7 >1.5 >1.5Poor peak shape and symmetry of E2

TEB, besides it showed very long retention time

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Table S3- Linearity of the developed method.

Analytes Concentration range (µmol L-1)

Linear equation r aANOVA Lack of Fit

F-value b p-value c

(+)-TEB 0.125 - 85.00 y = 0.1097x + 0.0099 0.9978 2.04 0.135(−)-TEB 0.125 - 85.00 y = 0.1079x + 0.0084 0.9993 2.57 0.075

(+)-TEBOH 0.005 – 0.900 y = 0.1352x – 0.0005 0.9993 1.92 0.152(−)-TEBOH 0.005 – 0.900 y = 0.1219x – 0.0005 0.9992 2.71 0.065

a correlation coefficientb Fcrit<Fcalc = 2.85c p > 0.05

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Table S4- Evaluation of matrix effect.

Analytes Nominal Concentration(μmol L-1) NMFa Precision

(%RSD)b

(+)-TEB 0.250 / 75.00 1.17 / 1.05 7 / 3

(−)-TEB 0.250 / 75.00 1.21 / 1.08 6 / 4

(+)-TEBOH 0.015 / 0.750 1.28 / 1.24 9 / 4

(−)-TEBOH 0.015 / 0.750 1.13 / 1.24 7 / 6a normalized matrix factor and b relative standard deviation

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Analyte Nominal Concentration(µmol L-1)

Obtained Concentration(µmol L-1)

Precision(%RSD)a

Accuracy(%RE)b

Intra-day

(+)-TEB 0.125 / 0.250 / 25.00 / 75.00 0.131 / 0.243 / 27.52 / 69.42 11 / 2 / 2 / 1 4.8 / -2.8 / 10.1 / 7.4

(−)-TEB 0.125 / 0.250 / 25.00 / 75.00 0.128 / 0.244 / 26.49 / 71.20 8 / 2 / 2 / 2 2.4 / 2.4 / 6.0 / 5.1

(+)-TEBOH 0.005 / 0.015 / 0.250 / 0.750 0.005 / 0.014 / 0.251 / 0.759 3 / 7 / 2 / 3 0.0 / -6.7 / 0.4 / 1.2

(−)-TEBOH 0.005 / 0.015 / 0.250 / 0.750 0.005 / 0.015 / 0.267 / 0.726 3 / 7 / 1 / 4 0.0 / 0.0 / 6.8 / -3.2

Inter-day

(+)-TEB 0.125 / 0.250 / 25.00 / 75.00 0.126 / 0.252 / 27.26 / 69.20 7 / 4 / 2 / 3 0.8 / 0.8 / 9.0 / -7.7

(−)-TEB 0.125 / 0.250 / 25.00 / 75.00 0.125 / 0.249 / 26.55 / 70.59 6 / 3 / 2 / 4 0.0 / -0.4 / 6.2 / -5.9

(+)-TEBOH 0.005 / 0.015 / 0.250 / 0.750 0.005 / 0.015 / 0.250 / 0.748 3 / 6 / 3 / 3 0.0 / 0.0 / 0.0 / -0.3

(−)-TEBOH 0.005 / 0.015 / 0.250 / 0.750 0.005 / 0.015 / 0.263 / 0.713 6 / 6 / 3 / 3 0.0 / 0.0 / 5.2 / -4.9

Table S5- Intra- and inter-day precision and accuracy.

a relative standard deviation and b relative error

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Table S6- Stability tests in different conditions.

Analyte Nominal Concentration(µmol L-1)

Obtained Concentration(µmol L-1)

Precision(%RSD)a

Accuracy(%RE)b

Incubation(30 min at 37ºC)

(+)-TEB 0.250 / 75.00 0.274 / 66.72 5 / 4 9.6 / -11.0

(−)-TEB 0.250 / 75.00 0.266 / 67.83 5 / 7 6.4 / -9.6

(+)-TEBOH 0.015 / 0.750 0.015 / 0.702 5 / 8 0.0 / -6.4

(−)-TEBOH 0.015 / 0.750 0.016 / 0.688 5 / 6 6.7 / -8.3

Autosampler(48 h at 10ºC)

(+)-TEB 0.250 / 75.00 0.259 / 72.77 4 / 4 3.6 / -3.0

(−)-TEB 0.250 / 75.00 0.249 / 73.64 7 / 6 -0.4 / -1.8

(+)-TEBOH 0.015 / 0.750 0.014 / 0.800 8 / 4 -6.7 / 6.7

(−)-TEBOH 0.015 / 0.750 0.015 / 0.751 6 / 4 -0.0 / 0.1a relative standard deviation and b relative error

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Fig. S1. Chemical structures of A) tebuconazole and B) 1-hydroxytebuconazole.

*Chiral center.

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Fig. S2. Circular dichroism of TEB and TEBOH enantiomers dissolved in methanol:

water (90:10, v/v). A) E1 TEBOH ((+)-TEBOH), B) E2 TEBOH ((−)-TEBOH), C) E1

TEB ((+)-TEB) and D) E2 TEB ((−)-TEB).

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Fig. S3. Screening procedure in A) polar organic mode (PO-HPLC) and B) reversed-phase mode (RP-HPLC)

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Fig. S4. Representative chromatograms of the enantioselective analysis of A) rac-

TEBOH, B) rac-TEB, and C) the mixture of rac-TEBOH and rac-TEB using a

Chiralcel OJ® column. 100% methanol as mobile phase, flow rate of 0.5 mL min -1,

column oven temperature of 20 ± 2ºC and detection at 220 nm. E1 TEBOH: first eluted

enantiomer of 1-hydroxytebuconazole; E2 TEBOH: second eluted enantiomer of 1-

hydroxytebuconazole; E1 TEB: first eluted enantiomer of tebuconazole; E2 TEB:

second eluted enantiomer of tebuconazole.

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Chiralcel OJ® column. Methanol: water (90:10, v/v) as mobile phase, flow rate of 0.5

mL min -1, column oven temperature of 20 ± 2ºC and detection at 220 nm. E1 TEBOH:

first eluted enantiomer of 1-hydroxytebuconazole; E2 TEBOH: second eluted

enantiomer of 1-hydroxytebuconazole; E1 TEB: first eluted enantiomer of

tebuconazole; E2 TEB: second eluted enantiomer of tebuconazole.

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Chiralcel OJ® column. Acetonitrile: water (60:40, v/v) as mobile phase, flow rate of 0.7

mL min -1, column oven temperature of 20 ± 2ºC and detection at 220 nm. E1 TEBOH:

first eluted enantiomer of 1-hydroxytebuconazole; E2 TEBOH: second eluted

enantiomer of 1-hydroxytebuconazole; E1 TEB: first eluted enantiomer of

tebuconazole; E2 TEB: second eluted enantiomer of tebuconazole.

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Peak 1= (+)-TEB and Peak 2= (−)-TEB

Fig S7. Racemization study at incubation conditions (phosphate buffer 100 mmol L-1,

pH 7.4, 0.2 mg mL-1 of HLM protein content and incubation for 20 minutes at 37ºC). A)

(−)-TEB (50 µmol L-1), B) (−)-TEB (2 µmol L-1), C) (+)-TEB (50 µmol L-1), D) (+)-

TEB (2 µmol L-1).

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