SUPLEMMENTARY MATERIAL In vitro enantioselective study of the toxicokinetic effects of chiral fungicide tebuconazole in human liver microsomes Maísa Daniela Habenschus 1 , Viviani Nardini 1 , Luís Gustavo Dias 1 , Bruno Alves Rocha 2 , Fernando Barbosa Jr 2 , Anderson Rodrigo Moraes de Oliveira 1 * 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 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1 2
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ars.els-cdn.com · Web viewresume the analytical conditions evaluated during the screening procedure for PO-HPLC and RP-HPLC, respectively. After the screening procedure with HPLC-DAD,
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SUPLEMMENTARY MATERIAL
In vitro enantioselective study of the toxicokinetic effects of chiral fungicide
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
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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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 - -
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Methanol: Ethanol 50:50 0.3 0 - -
Methanol: Isopropanol 85:15 0.3 0 - -
Ethanol 100 0.4 0 - -
Ethanol: Isopropanol 85:15 0.3 0 - -
Isopropanol 100 0.3 - - It was not possible to evaluate due to column high pressure
Chiralcel OD-H®
Acetonitrile 100 0.3 0 - -
Acetonitrile: Methanol 50:50 0.3 0 - -
Acetonitrile: Ethanol 85:15 0.3 0 - -
Acetonitrile: Isopropanol 50:50 0.3 0 - -
Methanol 100 0.3 0 - -
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Methanol: Ethanol 50:50 0.3 0 - -
Methanol: Isopropanol 50:50 0.3 0 - -
Ethanol 100 0.3 0 - -
Ethanol: Isopropanol 50:50 0.15 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
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