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SUPPLEMENTARY MATERIAL

Evaluation of the enantioselective in vitro metabolism of the chiral pesticide

fipronil employing a human model: risk assessment through in vitro-in vivo

correlation and prediction of toxicokinetic parameters

Daniel Blascke Carrão1, Isabel Cristina dos Reis Gomes1, Fernando Barbosa Junior2,

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: deoliveira@usp.br

Phone: +55-16-3315-3799

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Enantioselective HPLC method development

For the development of the enantioselective HPLC method, screening

procedures in the polar organic, reverse and normal phase modes [1] were accomplished

with chiral analytical columns to evaluate the separation of fipronil enantiomers. In the

polar organic mode, mobile phases comprising methanol, ethanol, acetonitrile,

isopropanol and their mixtures at different proportions were employed. In the reverse

phase mode, aqueous methanol or aqueous acetonitrile mixtures at different proportions

were used as mobile phases. Finally, in the normal phase mode, hexane: ethanol or

hexane: isopropanol mixtures at different proportions were evaluated as mobile phases.

In all the modes, the enantioselective separation was investigated with and without the

addition of acid and/or basic additives. The chiral columns consisted of the following

polysaccharide derivatives: Chiralpak AD-H® (150 mm x 4.6 mm, 5 μm), Chiralpak

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

Chiralcel OJ® (250 mm x 4.6 mm, 10 μm), all of which were acquired from Daicel

Chemical Industries (Tokyo, Japan); Lux Amylose-2® (150 mm x 4.6 mm, 5 μm), Lux

Cellulose-1® (150 mm x 4.6 mm, 5 μm), or Lux Cellulose-2® (150 mm x 4.6 mm, 5 μm),

all of which were purchased from Phenomenex (Torrance, CA, USA). Chiral columns

consisting of macrocyclic antibiotic derivatives such as Chirobiotic T®(150 mm x 4.6

mm, 5 μm), Chirobiotic TAG®(150 mm x 4.6 mm, 5 μm), and Chirobiotic V® (150 mm

x 4.6 mm, 5 μm), all of which were acquired from Sigma-Aldrich (St. Louis, MO, USA)

were also assessed. The separation of fipronil metabolites was further evaluated by

employing the best condition obtained for the separation of fipronil enantiomers.

Finally, slight adjustments in the chromatographic conditions were made to reduce the

analyses time without impairment the chromatographic resolution.

The main objective of the new enantioselective analytical method was to obtain

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an acceptable separation of fipronil enantiomers and their metabolites fipronil sulfone,

fipronil desulfynil, and fipronil sulfide, within the shortest time possible. To achieve

that, analytical screening strategies in the polar organic, reversed, and normal phase

modes were used.

After evaluation of all the chiral columns previously described, four analytical

conditions allowed the start of a separation or the separation of fipronil enantiomers: (i)

polar organic mode with Chiralcel OJ® column and isopropanol (100%) + 1% acetic

acid as the mobile phase at a flow rate of 0.2 mL min−1 (Figure S4A), (ii) reverse phase

mode with Chiralpak AD-H® column and acetonitrile/water (45:55, v/v) as the mobile

phase at a flow rate of 0.5 mL min−1 (Figure S4B), (iii) reverse phase mode with

Chiralpak AS® column and acetonitrile/water (40:60, v/v) as the mobile phase at a flow

rate of 0.8 mL min−1 (Figure S4C); and (iv) normal phase mode with Chiralcel OD-H®

column and hexane/isopropanol (90:10, v/v) as the mobile phase at a flow rate of 1.0

mL min−1 (Figure S4D). Based on Figure S4D, the normal phase mode and Chiralcel

OD-H® column were the most promising conditions to separate fipronil enantiomers.

However, when the metabolites were analyzed, the analysis lasted over 30 min (Figure

S5A). To shorten the analysis time, a gradient elution strategy was employed. Gradient

elution in normal phase HPLC separations separates chiral compounds within a shorter

time without impairing the chromatographic resolution [3]. The final analytical

condition for the enantioselective analysis of fipronil and its metabolites was obtained

by employing a Chiralcel OD-H® column and hexane/isopropanol (gradient elution

(v/v): 0.01 min – 10% isopropanol, 12.00 min – 30% isopropanol, 12.50 min – 10%

isopropanol, and 15.00 min – 10% isopropanol) as the mobile phase at a flow rate of 1.5

mL min−1. The analyses were performed at 30 °C, the injection volume was 50 μL, and

the analytes were detected at 280 nm. These parameters allowed separation of fipronil

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enantiomers and their metabolites in less than 15 min without impairing the resolution

(Figure S5B). Chiral stationary phases based on polysaccharides from amylose and

cellulose derivatives are the most commonly employed due to the wide diversity of

chiral compounds that these chiral selectors can separate [4–6]. Although the

enantioselective resolution of native polysaccharides is limited, their derivatization with

carbamates or aromatic esters increases chiral recognition, mostly when donor or

acceptor substituents are introduced in their phenyl portion [6,7]. The chiral selector

cellulose‐tris‐(3,5‐dimethylphenylcarbamate) has already been reported to separate most

chiral pesticides [8]. Regarding in vitro and in vivo enantioselective metabolism studies

of chiral pesticides, this stationary phase has been employed in 78% of the separations

[9]. The chiral recognition of chiral stationary phases consisting of carbamate

derivatives is related to interactions between the polar carbamate groups of the chiral

selector with the analytes [10]. The carbamate groups of the stationary phase interact

with the analytes through dipole-dipole forces involving the C=O moiety and through

hydrogen bonding involving the C=O and NH groups [10].

After fipronil enantiomers were isolated, they were individually injected into the

chromatograph under the chromatographic conditions described by Tan el. (2004) [11].

S-fipronil was the first enantiomer to elute, followed by R-fipronil. Bearing this elution

order in mind, the isolated enantiomers were injected under the conditions of the

developed method. S-fipronil was the first enantiomer to elute; R-fipronil was the

second. To ensure the reproducibility of the developed enantioselective analytical

method, a system suitability test was carried out. The results (Table S1) proved that the

method was reproducible; the relative standard deviation of the retention times was

below 2%. Compared to other literature methods that describe the enantioselective

separation of fipronil and its metabolites, the method developed here was faster.

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Therefore, normal phase enantioselective HPLC separation by gradient elution allowed

enantioselective evaluation of in vitro fipronil metabolism by HLMs with adequate

resolution and reproducibility within a relatively short analysis time.

Method validation

The enantioselective HPLC analytical method was validated according to the

Guideline on Bioanalytical Method Validation of the European Medicines Agency

(EMA) [2]. The evaluated parameters were linearity, selectivity, carryover, precision

and accuracy within and between-day, lower limit of quantification (LLOQ) and

stability. In addition, at this step racemization evaluation was also carried out. Linearity

was evaluated by spiking aliquots of 200 μL of microsomal medium with standard stock

solutions of rac-fipronil at final concentrations of 1.00, 3.00, 10.00, 25.00, 50.00, 75.00,

and 100.00 μmol L−1 for each enantiomer; fipronil sulfone at final concentrations of

0.10, 0.30, 1.00, 5.00, 10.00, 20.00 and 30.00 μmol L−1; fipronil desulfinyl at final

concentrations of 0.30, 1.00, 3.00, 5.00, 10.00, 20.00 and 30.00 μmol L−1; and fipronil

sulfide at final concentrations of 0.30, 1.00, 3.00, 5.00, 15.00, 20.00 and 30.00 μmol

L−1. The analytical curves were obtained by plotting the analyte/IS peak area ratio

versus the analyte concentration. Because the residual analysis of the analytical curves

showed a heteroscedastic behavior, the results were weighted by employing the 1/x2

factor. The linearity of the analytical curve was ensured by application of the ANOVA

lack-of-fit test with the Minitab 16 Statistical software (State College, PA, USA). The

method proved to be linear over 1.00–100.00 μmol for S- and R-fipronil, 0.10–30.00

μmol for fipronil sulfone, 0.30–30.00 μmol for fipronil desulfinyl, and 0.30–30.00 μmol

for fipronil sulfide (Table S2). The selectivity of the analytical method was determined

by analyzing the chromatograms of blank samples of HLMs (without the analytes or

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IS). Selectivity was achieved demonstrating that there were no interference peaks in the

retention time of fipronil enantiomers, their metabolites, or the IS (Figure S6).

Furthermore, the peak purity index was higher than 0.99 for all the analytes, which

confirmed that the wavelengths of the area of the spectrum to the right and to the left

were equal. The carryover effect was assessed by analyzing the chromatograms of blank

samples of HLMs after injection of a sample with calibration standards at the upper

limit of quantification of all the analytes. The presence of any matrix interference in the

retention time of the analytes or the IS and the carryover effect were considered

acceptable if the areas were less than 20% of the LLOQ area for the analytes and less

than 5% of the LLOQ area for the IS. No carryover effect was observed in the

chromatograms of the blank samples of HLMs after injection of the analytes at the

upper limit of quantification. Precision and accuracy were assessed by means of

analytical curves prepared on the same day as the samples. Precision was expressed as

relative standard deviation (% RSD), and accuracy was expressed as relative error (%

RE). Within-day precision and accuracy (n = 5) were evaluated for the LLOQ, low-,

medium-, and high-quality control samples of each analyte. Between-day precision and

accuracy (n = 3) of the same concentrations were determined on three different days.

Precision and accuracy were considered acceptable if % RSD ≤20 and % ER ±20 for

LLOQ, respectively, and % RSD ≤15 and % ER ±15% for other concentrations,

respectively. The results of within- and between-day precision and accuracy agreed with

the guideline requirements (Table S3). The LLOQ was 1.00, 1.00, 0.10, 0.30, and 0.30

μmol L−1 for S-fipronil, R-fipronil, fipronil sulfone, fipronil desulfinyl, and fipronil

sulfide, respectively; % RSD was lower than 7%, and % RE was lower than ±5% (Table

S3). The stability tests (n = 5) were conducted with two concentration levels of each

analyte. The analyte stability was evaluated under incubation conditions (at 37 °C for 60

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min) and in the equipment autosampler (at 22 ± 2 °C for 24 h). Stable samples were

quantified by employing analytical curves prepared on the same day. The samples were

considered stable if the determined concentration was ±15% of the nominal

concentration for each analyte. The stability test showed that fipronil enantiomers and

their metabolites were stable under the incubation conditions (37 °C for 60 min) and in

the equipment autosampler (22 ± 2°C for 24 h) (Table S4). Racemization (n = 5) was

evaluated by employing the isolated fipronil enantiomers at two concentrations levels

(2.00 and 60.00 μmol L−1). The samples were submitted to incubation conditions (37 °C

for 60 min), and the results were qualitatively analyzed by observing the conversion of

one enantiomer into the other. No racemization of fipronil enantiomers occurred under

the incubation conditions (37 °C for 60 min) at any concentration (Figure S7).

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REFERENCES

[1] A.A. Younes, H. Ates, D. Mangelings, Y. Vander Heyden, A Separation Strategy Combining Three HPLC Modes and Polysaccharide-based Chiral Stationary Phases, J. Pharm. Biomed. Anal. 75 (2013) 74–85. doi:10.1016/j.jpba.2012.11.019.

[2] EMA (European Medicines Agency), Guideline on Bioanalytical Method Validation, EMEA, Comm. Med. Prod. Hum. Use. 44 (2012) 1–23. doi:EMEA/CHMP/EWP/192217/2009.

[3] M.L. De la Puente, C.T. White, A. Rivera-Sagredo, J. Reilly, K. Burton, G. Harvey, Impact of Normal-phase Gradient Elution in Chiral Cromatography: A Novel, Robust, Efficient and Rapid Chiral Screening Procedure, J. Chromatogr. A. 983 (2003) 101–114. doi:10.1016/S0021-9673(02)01735-1.

[4] I. Matarashvili, I. Shvangiradze, L. Chankvetadze, S. Sidamonidze, N. Takaishvili, T. Farkas, B. Chankvetadze, High-performance Liquid Chromatographic Separations of Stereoisomers of Chiral Basic Agrochemicals with Polysaccharide-based Chiral Columns and Polar Organic Mobile Phases, J. Sep. Sci. 38 (2015) 4173–4179. doi:10.1002/jssc.201500919.

[5] M. Gegenava, L. Chankvetadze, T. Farkas, B. Chankvetadze, Enantioseparation of Selected Chiral Sulfoxides in High-performance Liquid Chromatography with Polysaccharide-based Chiral Selectors in Polar Organic Mobile Phases with Emphasis on Enantiomer Elution Order, J. Sep. Sci. 37 (2014) 1083–1088. doi:10.1002/jssc.201301318.

[6] B. Chankvetadze, Recent Developments on Polysaccharide-based Chiral Stationary Phases for Liquid-phase Separation of Enantiomers, J. Chromatogr. A. 1269 (2012) 26–51. doi:10.1016/J.CHROMA.2012.10.033.

[7] R. Geryk, K. Kalíková, J. Vozka, D. Plecitá, M.G. Schmid, E. Tesařová, Enantioselective Potential of Chiral Stationary Phases Based on Immobilized Polysaccharides in Reversed Phase Mode, J. Chromatogr. A. 1363 (2014) 155–161. doi:10.1016/j.chroma.2014.06.040.

[8] V. Pérez-Fernández, M.Á. García, M.L. Marina, Chiral Separation of Agricultural Fungicides, J. Chromatogr. A. 1218 (2011) 6561–6582. doi:10.1016/j.chroma.2011.07.084.

[9] N.C.P. de Albuquerque, D.B. Carrão, M.D. Habenschus, A.R.M. de Oliveira, Metabolism Studies of Chiral Pesticides: A Critical Review, J. Pharm. Biomed. Anal. 147 (2018) 89–109. doi:10.1016/j.jpba.2017.08.011.

[10] Y. Okamoto, Y. Kaida, Resolution by High-performance Liquid Chromatography Using Polysaccharide Carbamates and Benzoates as Chiral Stationary Phases, J. Chromatogr. A. 666 (1994) 403–419. doi:10.1016/0021-9673(94)80400-1.

[11] J. Tang, K.A. Usmani, E. Hodgson, R.L. Rose, In Vitro Metabolism of Fipronil by Human and Rat Cytochrome P450 and Its Interactions with Testosterone and Diazepam, Chem. Biol. Interact. 147 (2004) 319–329. doi:10.1016/j.cbi.2004.03.002.

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Figure S1. Fipronil solubility in microsomal medium (n = 3) without the addition of any solubilizing agent.

0 50000 100000 150000

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50

75

100

SampleControl

98% *

39% **

31% **

26% **

Peak area

[Fip

roni

l] (

mol

L-1

)

* p < 0.05** p > 0.05

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Figure S2. Fipronil solubility employing poloxamer 407 (0.05% m/v) as solubilizing agent in microsomal medium (n = 3).

0 50000 100000 150000

30

40

50

60

80

100

SampleControl

*

**

*

*

*

**

Peak area

[Fip

roni

l] (

mol

L-1

)

* p < 0.05** p > 0.05

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Figure S3. Influence of poloxamer 407 in fipronil metabolism by human CYP450 enzymes (n = 3).

0 25 50 75 100

Poloxamer 407

0.10 (% m/v)0.05 (% m/v)

CYP450 Remanescent Activity

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Figure S4. Representative chromatograms of the best analytical conditions for enantioselective separation of fipronil enantiomers.

A) Mode: polar organic, column: Chiralcel OJ®, mobile phase: isopropanol (100%) + 1% acetic acid, flow rate: 0.2 mL min−1, B) mode: reverse phase, column: Chiralpak AD-H®, mobile phase: acetonitrile: water (45:55, v/v), flow rate: 0.5 mL min−1, C) mode: reverse phase, column: Chiralpak AS®, mobile phase: acetonitrile: water (40:60, v/v), flow rate: 0.8 mL min−1 and D) mode: normal phase, column: Chiralcel OD-H®, mobile phase: hexane: isopropanol (90:10, v/v), flow rate: 1.5 mL min−1. (1) and (2) fipronil enantiomers. Rs: resolution.

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Figure S5. Representative HPLC chromatograms for the enantioselective analysis of fipronil and its metabolites.

(A) isocratic elution and (B) gradient elution. (1) S-fipronil, (2) R-fipronil, (3) fipronil sulfone, (4) fipronil desulfinyl, and (5) fipronil sulfide.

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Figure S6. Representative HPLC chromatograms for the enantioselective analysis of fipronil and its metabolites.

(A) HLMs spiked with fipronil, its metabolites and IS, (B) HLMs blank sample. (1) S-fipronil, (2) R-fipronil, (3) fipronil sulfone, (4) IS, (5) fipronil desulfinyl, and (6) fipronil sulfide.

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FIGURE S7. Racemization evaluation.

A) S-fipronil (2.00 µmol L−1); B) R-fipronil (2.00 µmol L−1); C) S-fipronil (60.00 µmol L−1), D) R-fipronil (60.00 µmol L−1). (1) – S-fipronil, (2) – R-fipronil, (3) – IS.

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Table S1. System suitability for the developed method.

Analytes Retention Time (min) Resolution Plate Numbers

S-fipronil 3.43 ± 0.2% - 1951 ± 4%

R-fipronil 4.12 ± 0.1% 2.1 ± 3% 2486 ± 5%

fipronil sulfone 6.36 ± 0.1% 6.2 ± 2% 4376 ± 3%

Internal standard 8.21 ± 0.1% 3.7 ± 1% 3034 ± 1%

fipronil desulfinyl 9.77 ± 0.1% 2.9 ± 1% 6821 ± 1%

fipronil sulfide 11.00 ± 0.1% 2.4 ± 1% 6698 ± 1%

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Table S2. Linearity for the developed method.

AnalytesConcentration Range(μmol L−1)

Linear Equation raANOVA Lack of Fit

F-valueb p-valuec

S-fipronil 1.00 – 100.00 y = 0.2187 x + 0.0481 0.9988 1.22 0.351

R-fipronil 1.00 – 100.00 y = 0.2252 x + 0.0583 0.9990 1.19 0.364

fipronil sulfone 0.10 – 30.00 y = 0.1663 x + 0.0036 0.9978 1.81 0.176

fipronil desulfinyl 0.30 – 30.00 y = 0.0879 x – 0.0009 0.9996 1.65 0.211

fipronil sulfide 0.30 – 30.00 y = 0.0015 x − 0.0005 0.9981 2.27 0.104a correlation coefficientb Fcrit<Fcalc = 2.79c p > 0.05

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Table S3. Within- and between-day precision and accuracy for the developed method.

AnalytesNominal Concentration(μmol L−1)

Obtained Concentration(μmol L−1)

Precision% RSDa

Accuracy % REb

Within-day

R-fipronil 1.00 / 3.00 / 50.00 / 75.00 0.99 / 2.88 / 49.64 / 74.50 6 / 3 / 3 / 4 −1 / −4 / −1 / −1

S-fipronil 1.00 / 3.00 / 50.00 / 75.00 0.98 / 3.07 / 49.33 / 73.37 4 / 4 / 4 / 2 −2 / 2 / −1 / − 2

fipronil sulfone 0.10 / 0.30 / 10.00 / 20.00 0.10 / 0.31 / 10.61 / 20.52 11 / 8 / 3 / 4 0 / 3 / 6 / 3

fipronil desulfinyl 0.30 / 1.00 / 10.00 / 20.00 0.30 / 0.99 / 9.88 / 20.12 7 / 3 / 7 / 3 0 / −1 / −1 / 1

fipronil sulfide 0.30 / 1.00 / 15.00 / 20.00 0.29 / 0.97 / 15.30 / 21.00 6 / 3 / 5 / 5 −3 / −3 / 2 / 5

Between-day

R-fipronil 1.00 / 3.00 / 50.00 / 75.00 1.00 / 2.85 / 48.55 / 74.90 7 / 7 / 4 / 4 0 / −5 / −3 / −0.1

S-fipronil 1.00 / 3.00 / 50.00 / 75.00 1.00 / 2.92 / 48.60 / 74.64 5 / 6 / 4 / 5 0 / −3 / −3 / −0.5

fipronil sulfone 0.10 / 0.30 / 10.00 / 20.00 0.10 / 0.31 / 10.38 / 21.15 9 / 8 / 4 / 4 0 / 3 / 4 / 6

fipronil desulfinyl 0.30 / 1.00 / 10.00 / 20.00 0.30 / 0.97 / 9.92 / 21.00 7 / 4 / 5 / 5 0 / −3 / −1 / 5

fipronil sulfide 0.30 / 1.00 / 15.00 / 20.00 0.30 / 0.95 / 15.29 / 21.45 8 / 6 / 5 / 4 0 / −5 / 2 / 7arelative standard deviationbrelative error

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Table S4. Fipronil and metabolites stability.

Analytes AnalytesNominal Concentration(μmol L−1)

Obtained Concentration(μmol L−1)

Precision% RSDa

Accuracy % REb

Incubation(60 min at 37°C)

S-fipronil 3.00 / 75.00 2.77 / 77.13 2 / 2 −8 / 3

R-fipronil 3.00 / 75.00 2.85 / 77.19 2 / 2 −5 / 3

fipronil sulfone 0.30 / 20.00 0.29 / 21.37 6 / 3 −3 / 7

Fipronildesulfinyl 1.00 / 20.00 1.01 / 21.71 8 / 4 1 / 9

fipronil sulfide 1.00 / 20.00 0.92 / 22.25 5 / 5 −8 / 11

Autosampler(24 h)

S-fipronil 3.00 / 75.00 2.88 / 77.58 2 / 6 −4 / 3

R-fipronil 3.00 / 75.00 2.85 / 77.19 4 / 6 −5 / 3

fipronil sulfone 0.30 / 20.00 0.29 / 21.95 7 / 7 −3 / 10

fipronil desulfinyl 1.00 / 20.00 0.97 / 21.59 6 / 4 −3 / 8

fipronil sulfide 1.00 / 20.00 0.94 / 21.55 3 / 5 −6 / 8arelative standard deviationbrelative error

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