Accepted Manuscript

Development and validation of an ultra performance liquid chromatography Q-

Exactive Orbitrap mass spectrometry for the determination of fipronil and its
metabolites in tea and chrysanthemum

Hongping Chen, Guanwei Gao, Pingxiang Liu, Meiling Pan, Yunfeng Chai, Xin
Liu, Chengyin Lu

PII: S0308-8146(17)31818-6
DOI: https://doi.org/10.1016/j.foodchem.2017.11.017
Reference: FOCH 21994

To appear in: Food Chemistry

Received Date: 12 April 2017

Revised Date: 26 October 2017
Accepted Date: 3 November 2017

Please cite this article as: Chen, H., Gao, G., Liu, P., Pan, M., Chai, Y., Liu, X., Lu, C., Development and validation
of an ultra performance liquid chromatography Q-Exactive Orbitrap mass spectrometry for the determination of
fipronil and its metabolites in tea and chrysanthemum, Food Chemistry (2017), doi: https://doi.org/10.1016/
j.foodchem.2017.11.017

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 Development and validation of an ultra performance liquid chromatography
Q-Exactive Orbitrap mass spectrometry for the determination of fipronil and its metabolites
in tea and chrysanthemum

Hongping Chen, Guanwei Gao, Pingxiang Liu, Meiling Pan, Yunfeng Chai, Xin Liu *,

Chengyin Lu *

Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, 310008, China

Key Laboratory of tea quality and safety, Ministry of Agriculture Hangzhou, 310008, China

*Corresponding author. Tel.: +86 571-86650607; fax: +86 571-86652004

E-mail address: Lchy@mail.tricaas.com (C. Lu)

Liuxin@mail.tricaas.com (X. Liu)

Abstract

A fast, sensitive and reliable method for the determination of fipronil and its metabolites in
tea and chrysanthemum was developed using a modified QuEChERS technique and an ultra
performance liquid chromatography Q-Exactive Orbitrap mass spectrometry. The mixture of
adsorbents containing primary secondary amine (PSA), octadecylsilane (C18) and carbon
nanotubes (CNTs), was used as QuEChERS adsorbents. The use of mass resolution at 70000 full
width at half maximum (FWHM) and narrow mass windows at 5 ppm achieved high selectivity
and repeatability. Satisfactory linearity with correlative coefficient (R2) higher than 0.996 was
achieved for all compounds. Recoveries at three levels (2, 10 and 50 µg kg-1) ranged from 86% to
112%, while the intra- and inter-day accuracies were less than 15%. Limits of quantification for
fipronil and its metabolites were 2 µg kg-1, which fulfils the requirement of maximum residue
limits formulated by European Union and Japan.

Keywords:
Tea; Fipronil; Metabolites; QuEChERS; Ultra performance liquid chromatography Q-Exactive
Orbitrap mass spectrometry
1. Introduction

Fipronil is a phenylpyrazole insecticide developed by Rhône-Poulenc Agro between 1985

and 1987 and placed on the market in 1993 (Ying, & Kookana, 2002). Initially, fipronil was
recommended to replace high toxic organophosphorus pesticides and extensively used in
agricultural crops against culture pests, clickbeetles, grasshopper ants, fleas due to its excellent
effect on pests control (Zhou, Lu, Liu, & Gan, 2004; Stevens, Helliwell, & Warren, 1998; Visscher,
1997; Michel, & Gilles, 1998). However, rising concerns have been voiced about its human health
and environmental effects (Tingle, Rother, Dewhurst, Sasha, & William, 2013). It has been known
that fipronil was characterized with elicit neurotoxicity via an interaction with ionotropic receptors,
namely GABA and glutamate receptors (Vidau, González-Polo, Gómez-Sánchez, Bravo-San
Pedro, Pizarro-Estrella, Blasco, Brunet & Fuentes, 2011). High ecological risk of fipronil was also
attracted global attention due to its high toxicity of birds, vertebrates, and aquatic animals (EPA,
2001). In addition, fipronil could be degraded by means of oxidation, reduction, hydrolysis and
photolysis to form three major products: fipronil sulphone, fipronil sulfide and fipronil desulfinyl
(Bobé, Meallier, Cooper, & Coste, 1998). Previous studies have shown that fipronil metabolites
possessed longer persistence (Xavier, Chandran, George, Beevi, Methew, Paul, Arimboor,
Vijayasree, Pradeepkumar, & Rajith, 2014; Schlenk, Huggett, Allgood, Bennett, Rimoldi, Beeler,
Block, Holder, Hovinga, & Bedient, 2001) and more potent endocrine activity than the parent
compounds (Lu, Du, Zhou, Chen, Lu, & Zhang, 2015; Cravedi, Delous, Zalko, Viguié, &
Debrauwer, 2013). Residue of fipronil and its metabolites in crops and environment can be
transferred to foods and contribute to the dietary exposure of a consumer. Recently, restriction or
forbiddance of the use of fipronil in crops has been formulated with strict maximum residual
limits (MRLs). MRLs of fipronil and its metabolites in tea set by European Union and Japan is
0.005 mg kg-1 and 0.002 mg kg-1, respectively (European Commission EU pesticides Database,
2015; The Japan Food Chemical Research Foundation,2015). In China, registration of fipronil in
crops except maize has been forbidden since 2009 (Ministry of Agriculture of the People’s
Republic of China. 2015). Therefore, determination of fipronil residue is a challenge due to its
requirement of extremely low limit of detection.
Fipronil analysis from tea samples has appeared in published multi-residue methods, which
focused on high throughout and sample efficiency. In these cases, pesticide recoveries and/or
limits of detection (LODs) are not optimal. For fipronil multi-residue methods for tea, the LODs
were over 10 µg kg-1 (Pang, Fan, Zhang, Li, Chang, Cao, Liu, Li, Wang, Hu, & Liang, 2011; Chen,
Yin, Wang, Jiang, & Liu, 2014; Zhang, Mobley, Zhang, Zheng, Lu, Ragin, & Smith, 2010; Hou,
Lei, Qiu, Guo, Yi, & liu, 2014) , which is higher than its MRLs in tea and could not fulfill the
requirement. Reducing LODs could be solved by using high sensitive analytical instrument or
injecting larger volume of sample liquids into analytical instrument (Wu, Yu, Xu, Dong, Liu, Du,
Wei, & Zheng, 2017). However, more samples could result in serious matrix effect and
contamination for analytical instruments.
Recently, liquid chromatography Q-Exactive Orbitrap high solution mass spectrometry has
been widely used for the multi residue analysis of pesticides and it was found to be increasingly
importance for identification, qualitative screening and quantitative analysis of pesticides and their
metabolites (Lin, Liu, Hu, Liu, & Ruan, 2016; Barnaba, Dellacassa, Nicolini, Nardin, Malacarne,
& Larcher, 2015; Yang, Li, Wang, & Liu, 2016. The Orbitrap mass analyzer was firstly introduced
in 2000 (Makarov, 2000). The use of the Orbitrap mass analyzer preceded by an external injection
device of the C-trap allows high-accuracy mass measurements within 2 parts per million (ppm)
using internal standard and 5 ppm with external calibration (Makarov, Denisov, Kholomeev,
Balschun, Lange, Strupat, & Horning, 2006). Compared with triple MS and TOF MS, the Orbitrap
high solution mass has higher sensitivity in full scan mode, higher sensitivity and higher intensity
range (Hogneboom, Leerdam, & Voogt, 2009). Therefore, Orbitap MS was considered as a
powerful technique for the analysis of novel chemicals, degraded products and metabolites
(Hamide, Vural, & Ebru, 2015).
Taking into account the shortage of the present analytical methods, this study is aimed to
develop a sensitive method for the determination of fipronil and its metabolites to fulfill the
requirement of maximum residue limits (MRLs) formulated by European Union and Japan. A
modified QuEChERS technique was optimized for simple and fast sample preparation. A powerful
ultra performance liquid chromatography coupled with Q-Exactive Orbitrap mass spectrometry
(UPLC Q-Exactive Oribtrap MS) system with accurate mass full scan was employed to detect
fipronil and its metabolites.

2. Experimental

2.1. Reagents and chemicals

Fipronil (98.5%), fipronil desulfinyl (99%), fipronil sulphide (99.5%) and fipronil sulphone
(99%) were purchased from Dr. Ehrenstorfer GmbH Company (Augsburg, Germany). HPLC
grade of acetonitrile, and methanol were obtained from Sigma-Aldrich (Shanghai, China). Water
was purified by the Milli Q system (Millipore, Miford, USA). Analytical-grade sodium chloride
and anhydrous magnesium were purchased from Zhejiang Medicine (Hangzhou, China) and were
baked at 650 °C for 4 h prior to use. Dispersive adsorbents of primary secondary amine (PSA),
graphitized carbon black (GCB), carbon nanotubes (CNTs), octadecylsilane (C18), florisil, strong
anion exchange (SAX), and strong cationic exchange (SCX) were provided by Bonna-Agela
Technologies (Tianjin, China).

2.2. Sample preparation

Previously grounded tea (2.5 g) was weighed into a 50-mL centrifuged tube. Fipronil and its
metabolites were added to concentrations of 2, 10 and 50 µg/kg for recovery test. A volume of 5
mL water was added to each sample. The mixture was mixed with a vortex and allowed to sock
for 30 min. After that, 10 mL acetonitrile was used to extract the target compounds by vigorously
mixing for 2 min using a vortex. Then, 4 g anhydrous MgSO4 and 2 g NaCl were added into the
centrifuged tube. Each sample was immediately mixed with a vortex for 2 min, and centrifuged at
16525 g for 10 min. One mL of the upper acetonitrile layer was transferred into 2-mL centrifuged
tube containing 200 mg PSA, 100 mg C18 and 10 mg CNTs. The mixture was mixed using a
vortex for 2 min and then centrifuged 16525 g for 10 min. The upper layer was transferred into
autosampler vial through 0.22 µm filter membrane.

2.3. Preparation of standard solutions

Stock standard solution of fipronil, fipronil desulfinyl, fipronil sulphide and fipronil sulphone
at 1000 µg/mL was prepared by accurately weighing solid standards and being dissolved in
methanol. Mixture of working standard solution at 10 µg mL-1 was prepared in methanol. Matrix
matched calibration solutions were prepared by the dilution of working standard solutions using
blank sample extracts obtained from organic tea with the treatment referred with the section “2.2.
Sample preparation”. All standard solutions were stored in -4 oC and matrix matched calibration
standard solution was immediately analyzed.

2.4. Instrumentation conditions

The chromatographic analysis of fipronil and its metabolites was performed using an
Ultimate 3000 UPLC system (Thermo Fisher Scientific, San Jose, CA, USA). Separations were
carried out on a Hypersil GOLD C18 analytical column (100 × 2.1 mm, 3 µm) from Thermo Fisher
Scientific (San Jose, CA, USA). The mobile phase consisting (A) water and (B) methanol was
delivered at the flow rate of 0.3 mL min−1. The elution gradient condition initially set up 10% B,
and rose to 50% B in 3 min, then increased 100% B in 5 min and held for 7 min. Finally, the
mobile phase was returned to initial condition. The injection volume was 5 µL.
The UHPLC system was coupled to a Q-Exactive Orbitrap MS (Thermo Fisher Scientific,
Bremen, Germany) operating with a heated electrospray interface (HESI) in negative electrospray
ionization (ESI-). The temperature of ion transfer capillary, spray voltage, sheath gas flow rate,
auxiliary gas flow rate and S-lens RF level were set to 325 o C, 3.5 kV, 60, 30 and 55, respectively.
The Q-Exactive MS was tuned and calibrated in positive and negative mode once a week using the
calibration solutions, including caffeine, MRFA, and a mixture of fluorinated phosphazines
ultramark 1621. The mass spectra were acquired with full MS mode at a resolution of 70000
FWHM with 2.0 × 106 of Automatic Gain Control (AGC) target and 100 ms of maximum ion
injection time. The analyses were performed without lock mass. Data were processed using
Xcalibur™ version 2.2.1 and Trace Finder 3.3 version (Thermo Fisher Scientific, Les Ulis,
France).

2.5. Validation procedure

The specificity was assessed by preparing and analyzing four varieties of blank tea samples
(green tea, black tea, oolong tea and puerh tea) and chrysanthemum. Linearity was investigated by
analysis of the calibration curves over the concentration ranges of 0.5–100 ng mL-1 using
least-squares linear regressing of peak area versus concentration. The limits of quantification
(LOQs) were estimated based on the lowest concentration measurable with precision and accuracy
less than 20%. The precision and accuracy of the method were evaluated by analyzing the blank
samples spiked with standard solutions at 2, 10 and 50 µg kg-1.

3. Results and discussion

3.1 Optimization of UPLC Q-Exactive Orbitrap MS

Buffer salt has the effect on ionization of target compounds at ESI source, resulting in
increasing or reducing sensitivity of pesticides detected by mass spectrometry (Flores,
Romero-González, Frenich, & Vidal, 2012) . We tseted four systems including water, 0.1% formic
acid, 2 mM ammonium fromate, and 0.1% formic acid plus 2 mM ammonium formate. The results
are shown in Fig 1, and water was selected as mobile phase because higher sensitivity was
obtained for fipronil and its metabolites.
 Generally, UPLC Q-Exactive Orbitrap MS with full MS and full MS/dd-MS2 mode was used
to qualitative screening and quantitative analysis although several monitoring modes were
provided, including all ions fragmentation (AIF), full MS/AIF, targeted-selected ion monitoring
(SIM), parallel reaction monitoring (PRM), targeted-SIM/dd-MS2, full MS/AIF/natural loss
(NL)-dd-MS2 (Cai, Yang, Chen, Zhou, & Hong, 2016; Barnaba, Nardin, & Pietti, 2017; Roca,
Leon, Pastor, & Yusà, 2014). For full MS/dd-MS2 mode, full scan in the range m/z 100-1000 was
firstly carried out, and then these ions with high intensity threshold higher than 20000 were further
fragmented to obtain MS2 ions. Full MS/dd-MS2 mode provides more fragmentation ions
information, which is beneficial for identification and conformation of unknown or targeted
compounds (López-Gutiérrez, Romero-González, Frenich, & Martínez, 2014; Shi, Guo, Gong, Li,
Dong, Zhang, Cui, Jiang, Zhao, & Zeng, 2014). However, full MS/dd-MS2 should take scanning
time to monitor the MS2 ions and therefore lose the sensitivity. Fig. 2 shows the UPLC Q-Exactive
Orbitrap MS chromatograms of fipronil and its metabolites at 1 ng mL-1, obtained from full
MS/dd-MS2 and full MS mode. Intensity of fipronil and its metabolites at full MS mode was
4.7-10.8 times higher than that at full MS/dd-MS2. Therefore, UPLC Q-Exactive Orbitrap MS at
full MS mode was selected to achieve high sensitivity.
For high resolution mass spectrometry, two key parameters, e.g. resolution and extracted
mass tolerance, have an important effect on qualitative and quantitative analysis. False positive
detections can be occurred when too broad extracted mass tolerance sets for
extracted-ion-chromatogram construction, while false negative detection can also be occurred
when mass accuracy shift or too narrow extracted mass tolerance results in the monitored ions
outside the mass-extracted-window (Glauser, Grund, Gassner, Menin, Henry, Bromirski, Schütz,
Mcmullen, & Rochat, 2016). In this study, extracted mass tolerance of 2-20 ppm was evaluated by
extracted ion chromatograms, and symmetric chromatographic peaks of fipronil and its
metabolites were obtained with the extracted mass tolerance of higher than or equal to 5 ppm (Fig
S-1). Higher resolution helps to improve selectivity and avoid false detection, whereas fewer
points per peak can be obtained because ions have to spend more time in the Q-Exactive Orbitrap
analyzer and the time between consecutive analyses is longer. Resolutions of 17500, 35000, and
70000 were corresponded with 12, 7 and 3 scans per second (Rajski, Gómez-Ramos, &
Fernández-Alba, 2014). Enough scans (higher than 40 scans) per peak can be achieved for
resolutions of 17500, 35000, 70000 and 140000, and no interferential peaks were observed (Fig
S-2). Finally, resolution of 70000 was used in this study. Table 1 demonstrated the theoretical and
experimental masses for each analyte.

3.2 Optimization of modified QuEChERS

Seven adsorbents (GCB, CNTs, C18, PSA, SAX, SCX and florisil) were evaluated for the
effect of cleanup and recoveries of fipronil and its metabolites with modified QuEChERS
treatment. Firstly, each adsorbent with different mass was individually added in the mixture (1 mL)
of fipronil and its metabolites standards at 20 ng/mL. The solutions were mixed with a vortex for 2
min, and then centrifuged at 16525 g for 10 min. For CNTs, low mass was investigated at 5,10, 25
and 50 mg/mL, while the mass of 10, 25, 50 and 100 mg/mL was evaluated for GCB. For PSA,
C18, SAX, SCX and florisil, the mass of 50, 100, 150 and 200 mg/mL were investigated. The
results indicated that two adsorbents (CNTs and SCX) resulted in the loss of fipronil and its
metabolites, while others have no adsorbability of fipronil and its metabolites. The use of CNTs at
50 mg/mL resulted in the loss of fipronil and its metabolites about 5-13%, while SCX with the
mass at 100-200 mg/mL resulted in the loss of four target compounds in the range of 20-63%.
Furthermore, the raw extracts obtained from green tea were used to investiage the cleanup effect
with different volume of seven adsorbents. The extracts were light yellow and transparent when
CNTs at 10-50 mg/mL or GCB at 50-100 mg/mL were used. Purification effect were not obvious
for PSA, SAX, SCX, C18 and florisil. Compared with GCB, CNTs have the higher adsorbality of
the same compounds, such as pigments and organic molecules. Thus, 10 mg of CNTs was selected
as one of modified QuEChERS adsorbents. Our previous study showed that PSA and C18 have the
adsorbality of tea polyphenols and caffee (Chen, Yin, Wang, Jiang, & Liu, 2014). In this study, we
choose the mixture of CNTs (10 mg), PSA (200 mg) and C18 (100 mg) as the modified
QuEChERS adsorbents. Finally, the proposed adsorbents were used to evaluate the cleanup effect
and recoveries of fipronil and its metabolites. Satisfied recoveries of 4 target compounds with the
range of 97-112% were obtained for green tea, black tea, oolong tea, puer tea and chrysanthemum.
The extracts were light yellow and transparent for tea samlpes and chrysanthemum.

3.3 Matrix effect and linearity

Matrix effect was evaluated by comparing the slopes of calibration curves obtained from
matrix-matched calibration solutions and standard solutions prepared with methanol in the range
of 0.5 to 100 ng mL-1. Matrix effect (ME, %) was calculated by the equation: ME =
(Slopem/Slopes -1)×100, where Slopem is the slope of calibration plot with matrix-matched
calibration solutions, Slopes is the slope of calibration plot with solvent calibration solutions.
Matrix supression (values below -20%) was oberseved for fipronil and its metabolites in
chrysanthemum, and fipronil sulfide in green tea, and fipronil in oolong tea, black tea and puer tea,
which are fermentation tea (Table 2). Therefore, matrix matched calibration solutions were used to
avoid matrix effect for quantification purpose.
Linearity was evaluated by least squares linear regression analysis of chromatographic peak
areas vs concentrations in the range of 0.5-100 ng mL-1 preparing by blank samples (four types of
tea samples and chrysanthemum). As shown in Table 2, the determined corelation coefficients (R2)
were higher than 0.996 and each deviation of individual plot for calibration was lower than 20%.

3.4 Method Validation

Trueness was estimated by recovery test at three levels (2, 10 and 50 µg kg-1) in order to
fulfill the requirement of MRLs of fipronil residue in tea formulated by European Union and
Japan. Table 3 summarizes the results and Figure 3 shows the UPLC Q-Exactive Orbitrap MS
chromatograms obtained from green tea matrix matched calibration solutions at 0.5 ng mL-1,
spiked samples at 2 µg kg-1, and blank sample. Recoveries ranged from 86% to 112% for fipronil
and its metabolites spiked in tea samples and chrysanthemum. The results indicated that this
developed method was satisfactory for the analysis of fipronil and its metabolites in tea samples
and chrysanthemum.
Precision was evaluated by performing intra- and inter-day repeatability studies. As shown in
Table 3, repeatability values for relative standard deviations (RSDs) and interday precisions were
below 15%, indicating stability of this proposed method.
LODs were estimated analyzing three blank samples spiked at 0.1, 0.2, 0.5, 1.0, and 1.5 µg
-1
kg . They are defined as the minimum concentration at which the accurate mass error was <5 ppm.
LODs are as follows: fipronil 0.5 µg kg-1 for tea samples and 1.0 µg kg-1 for chrysanthemum,
fipronil desulfinyl and fipronil-sulphide, 1.0 µg kg-1 for tea samples and 1.5 µg kg-1 for
chrysanthemum, fipronil sulphone 0.2 µg kg-1 for tea samples and 0.5 µg kg-1 for chrysanthemum.
Limits of quantifications (LOQs) of fipronil and its metabolites in tea samples and
chrysanthemum were defined as the lowest spiked level, which was 2 µg kg-1.

3.5 Method Application

The developed method was employed to analyze 30 green tea samples, 10 black tea samples,
30 oolong tea samples, 10 puer tea samples and 20 chrysanthemum samples, which were obtained
in 2009-2016 from Chinese supermarkets, monopoly stores, and wholesale markets. The results
were illustrated in Table S-1. Frequency of fipronil and its metabolites in green tea, black tea,
oolong tea, puer tea and chrysanthemum was 20%, 40%, 56.7%, 10% and 50%, respectively.
Higher residues of fipronil and its metabolites were found in oolong tea and chrysanthemum,
which of maximum detected concentrations were 600.3 and 169.1 µg kg-1, respectively. Fipronil
and its metabolites were detected exceeding MRLs in tea formulated European Union (5 µg kg-1)
by 16.7%, 46.7%, 30%, 0 and 50% for green tea, black tea, oolong tea, puer tea and
chrysanthemum, respectively. The rate of simultaneous detection of fipronil and three metabolites
in positive samples was 51.4%, while 24.3% of positive samples only contained fipronil without
its metabolites. Due to toxicity of fipronil metabolites (Tavares, Palma, Medeiros, Santan, &
Mingatto, 2015; Gunasekara, Truong, Goh, Spurlock, & Tjeerdema, 2007), fipronil and its
metabolites should be simultaneously detected and evaluated for tea safety.
Furthermore, this developed method was also used to evaluate the transfer rates of fipronil
and its metabolites during green tea brewing. A green tea contained fipronil 17.5 µg kg-1, fipronil
desulfinyl 27.6 µg kg-1, fipronil sulphide 6.6 µg kg-1, and fipronil sulphone 7.8 µg kg-1, was used to
tea brewing. Brewing method was referred to our previous study (Chen, Pan, Pan, Zhang, Liu, &
Lu, 2016). Liquid-liquid extraction (LLE) with dichloromethane was employed to extract fipronil
and its metabolites from tea infusion. Dichloromethane was evaporated into dryness, and then the
extracts was dissolved by 2 mL methane for UPLC Q-Exactive Orbitrap MS analysis. Transfer
rates of fipronil, fipronil dessulfinyl, fipronil sulphide and fipronil sulphone were 10.4%±0.5%,
17.3%±1.6%, 10.4%±0.5%, 8.3%±0.6% and 12.1%±0.3%, respectively. Figure S-3 shows the
chromatograms of fipronil and its metabolites in green tea, first infusion and second infusion,
which indicated that transfer rates were similar between the first infusion and second infusion.

4. Conclusion

In this study, a fast, reliable and sensitive analytical method based on modified QuEChERS
followed by UPLC Q-Exactive Orbitrap MS was developed and optimized for simultaneous
determination of fipronil and its metabolites in tea and chrysanthemum. A mixture of adsorbents
containing 200 mg PSA, 100 mg C18 and 10 mg carbon nanotubes was used to purify the extracts.
High sensitivity and selectivity were obtained using UPLC Q-Exactive Orbitrap MS in negative
ESI ionization and full MS mode. The high sensitivity of this analytical methodology fulfills the
requirement of fipronil MRLs in tea formulated by European Union and Japan.
This developed method was used to survey fipronil and its metabolites residue in tea samples
and chrysanthemum, as well as their transfer rates during green tea brewing. Fipronil combination
with its metabolites was always simultaneously detected, indicating that total residues of fipronil
and its metabolites should monitored. Transfer rates of fipronil and its metabolites were
8.3%-17.3% during green tea brewing.

Supplementary materials
Three figures (Fig S-1, S-2 and S-3) and one table were shown in Supporting Information.

Acknowledgements
This work was supported by the earmarked fund for National Natural Funding (31671941),
Innovative Research Team in Chinese Academy of Agricultural Sciences
(CAAS-ASTIP-2014-TRICAAS), Public Welfare and Technique Project of Zhejiang province
(2017C32059) and Modern Agro-Industry Technology Research System (CARS-23),

Notes
The authors declare no competing financial interest.

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Figure Captions:
Fig. 1
Chromatographic peak area values of fipronil and its metabolites (at 10 ng/mL) obtained with
UPLC mobile phase of methanol with water, 0.1% formic acid, 2 mM ammonium formate or 0.1%
formic acid plus 2 mM ammonium formate
Note, F: Fipronil

Fig. 2
UPLC Q-Exactive Orbitrap MS chromatograms of fipronil and its metabolites at 1 ng mL-1,
obtained from full MS/dd-MS2 (A) and full MS mode (B)

Fig.3
UPLC-Orbitrap MS chromatograms of fipronil, fipronil dessulfinyl, fipronil sulphide and fipronil
sulphone in green tea matrix matched calibration solution (A) at 0.5 ng mL-1, green tea spiked at 2
µg kg-1 (B), and blank green tea (C).
Fig. 1

3.0E+08

2.0E+08

1.0E+08

0.0E+00
Fipronil F-desulfinyl F-sulphide F-sulphone

water
0.1% formic acid
2 mM ammonium formate
2 mM ammonium formate+0.1% formic acid
Fig. 2
RT: 0.00 - 15.00 SM: 15G
6.14 NL:
100 7.25E5
6.11
Fipronil A m/z=
50 434.9290-
434.9334
MS STD 1ppb
1.06 1.68 3.85 4.67 6.33 9.42 10.59 13.74
Relative Abundance
0 NL:
6.10
100 5.49E5
Fipronil desulfinyl m/z=
50 386.9625-
386.9663
MS STD 1ppb
3.15 4.41 5.00 6.20 8.07 10.15 10.94 11.65 13.14
0 NL:
6.15
100 6.18 3.68E5
m/z=
Fipronil sulphide 418.9345-
50
418.9387
MS STD 1ppb
1.93 2.92 3.94 6.38 10.15 13.88
0 NL:
6.22
100 2.67E6
m/z=
50 Fipronil sulphone 450.9241-
450.9287
MS STD 1ppb
0.45 2.39 3.20 4.61 6.37 7.81 9.78 12.00 14.69
0
0 2 4 6 8 10 12 14
Time (min)

RT: 0.00 - 15.00 SM: 15G

6.13 NL:
100 B 4.12E6
m/z=
50 434.9290-
434.9334
MS STD 1ppb
0.68 2.70 4.04 7.77 11.19 13.70
Relative Abundance

0 NL:
6.09
100 4.26E6
m/z=
50 386.9625-
386.9663
MS STD 1ppb
1.11 3.41 4.74 10.02 13.31
0 NL:
6.17
100 3.98E6
m/z=
50 418.9345-
418.9387
MS STD 1ppb
1.46 3.62 4.89 7.17 8.52 13.52
0 NL:
6.22
100 1.26E7
m/z=
50 450.9241-
450.9287
MS STD 1ppb
0.70 2.48 4.11 5.35 8.02 9.55 13.90
0
0 2 4 6 8 10 12 14
Time (min)
Fig.3
RT: 0.00 - 15.00 SM: 15G
6.13 NL:
100 2.25E6
m/z= A
50 Fipronil 434.9290-
434.9334 MS
GTJZSTD
0.48 8.31 13.72 0-5ppb
Relative Abundance 0
6.09 NL:
100
1.52E6

50
Fipronil desulfinyl
m/z=
386.9625-
386.9663 MS
0.58 11.83 13.40 GTJZSTD
0 0-5ppb
6.16
100 NL:
1.70E6
Fipronil sulphide
m/z=
50
418.9345-
418.9387 MS
0.78 6.27 14.76 GTJZSTD
0
6.22 0-5ppb
100
NL:
5.06E6
50 Fipronil sulphone
m/z=
450.9241-
0.49 11.96 13.19 450.9287 MS
0 GTJZSTD
0 2 4 6 8 10 12 14 0-5ppb
Time (min)

RT: 0.00 - 15.00 SM: 15B

6.14 NL:
100 1.79E6
m/z=
B
434.9290-
50
434.9334 MS
GTQC3_2-
0.05 2ppb
Relative Abundance

0
6.09 NL:
100
1.26E6
m/z=
50 386.9625-
386.9663 MS
11.43 12.55 GTQC3_2-
0 2ppb
6.17
100 NL:
1.31E6
50 m/z=
418.9345-
418.9387 MS
0.65 14.02 GTQC3_2-
0
6.22 2ppb
100
NL:
4.19E6
50 m/z=
450.9241-
0.43 8.67 12.10 14.93 450.9287 MS
0 GTQC3_2-
0 2 4 6 8 10 12 14 2ppb
Time (min)

RT: 0.00 - 15.00 SM: 15G

0.82 NL:
100 7.86E3
m/z=
C
434.9290-
50
434.9334
MS blank
Relative Abundance

0 NL:
9.75 13.86
100 9.91 1.92E4
11.15
m/z=
9.65 386.9625-
50 11.59
386.9663
MS blank
0 NL:
100 0
m/z=
50 418.9345-
418.9387
MS blank
0 NL:
0.81 13.83
100 7.90E3
m/z=
50 450.9241-
450.9287
MS blank
0
0 2 4 6 8 10 12 14
Time (min)
Table 1 Molecular, retention time, accurate mass, mass errors of fipronil and its metabolites
Retention Deprotonated ion
Molecular Mass error
Compound Time
formula Theoretical Experimental (ppm)
(min)
Fipronil C 12H 4C l2F6N 4OS 6.12 434.9309 434.9312 0.7
Fipronil-
C 12H 4C l2F6N 4 6.08 386.9639 386.9644 0.1
desulfinyl
Fipronil-sulphide C 12H 4C l2F6N 4S 6.15 418.9359 418.9366 0.2
Fipronil- sulphone C 12H 4C l2F6N 4O2S 6.20 450.9258 450.9264 0.1
Table 2 Linear equation, correlation coefficent and matrix effect of fipronil and its metabolites
Linear equation (R2) ME,%
solvent GT BT OT PT Mum G B O P M
T T T T um
F Y=1.823 Y=1.914 Y=1.154 Y=1.115 Y=1.257 Y=4.168 5 - - - -77
×107 x ×107x ×107x ×107x ×107x ×106x 3 3 3
+2.119× +1.645× +2.092× +6.740× +1.325× +1.297× 7 1 1
107 107 107 107 107 106
(0.9995) (0.9986) (0.9962) (0.9998) (0.9993) (0.9999)
F-d Y=1.203 Y=1.101 Y=1.255 Y=1.210 Y=1.391 Y=4.967 - - 1 1 -59
es ×107 x ×107x ×107x ×107x ×107x ×106x 8 4 6
+6.321× +1.777× +3.027× +2.092× +2.530× +3.729×
107 107 107 107 107 106
(0.9996) (0.9987) (0.9961) (0.9983) (0.9981) (0.9990)
F-fi Y=1.823 Y=1.165 Y=1.945 Y=1.838 Y=1.999 Y=7.673 - 7 1 1 -58
de ×107 x ×107x ×107x ×107x ×107x ×106x 3 0
+2.119× +3.210× +3×107 +1.499× +6.806× +2.852× 6
107 107 (0.9983) 107 107 106
(0.9995) (0.9962) (0.9998) (0.9997) (0.9998)
F-f Y=3.381 Y=3.432 Y=3.597 Y=3.564 Y=3.991 Y=2.264 2 6 5 1 -33
one ×107 x ×107x ×107x ×107x ×107x ×107x 8
+2.907× +4.971× +6.787× +3.894× +8.647× +1.618×
107 107 107 107 107 107
(0.9986) (0.9983) (0.9970) (0.9996) (0.9973) (0.9998)
GT: green tea, BT: black tea, OT: oolong tea, PT: puer tea, Mum: Chrysanthemum

Table 3 Recoveries (%) and repeatability values (relative standard deviations, %; inter-day
repeatability values, %) of fipronil and its metabolites in fortified tea samples and chrysanthemum
at 2, 10 and 50 µg/kg (n=5)
Compou Green tea black tea oolong tea Puer tea chrysanthemu
nds m
2 10 50 2 10 50 2 10 50 2 10 50 2 10 50
μg μg μg μg μg μg μg μg μg μg μg μg μg μg μg
kg- kg- kg- kg- kg- kg- kg- kg- kg- kg- kg- kg- kg- kg- kg-
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Fipronil 95 97 99 97 96 95 93 95 97 86 10 10 97 11 96
(3. (2. (1. (2. (1. (1. (3. (2. (1. (5. 5 3 (7. 2 (3.
5) 1) 7) 6) 9) 1) 6) 0) 3) 2) (2. (1. 9) (6. 5)
(8. (3. / (8. (1. / (7. (1. / (7. 2) 2) (14 3) /
6)* 7) 4) 6) 5) 5) 3) (1. / .8) (9.
4) 4)
Fipronil- 91 93 99 10 10 10 10 96 10 88 93 10 94 93 10
desulfiny (3. (1. (1. 6 4 5 2 (2. 2 (4. (1. 0 (6. (5. 2
l 7) 9) 1) (3. (2. (1. (4. 9) (1. 8) 6) (2. 6) 3) (2.
(9. (4. / 9) 3) 6) 8) (2. 3) (8. (6. 0) (11 (9. 9)
6) 3) (5. (3. / (5. 1) / 5) 7) / .2) 7) /
1) 4) 2)
Fipronil- 91 88 98 97 10 10 10 99 98 99 95 97 97 95 93
sulphide (3. (2. (3. (4. 2 6 7 (4. (1. (4. (1. (2. (7. (4. (3.
6) 7) 2) 6) (0. (1. (5. 3) 5) 6) 1) 3) 1) 1) 6)
(11 (6. / (6. 8) 3) 5) (5. / (9. (4. / (10 (6. /
.5) 3) 8) (4. / (6. 3) 8) 7) .5) 8)
2) 4)
Fipronil- 90 96 93 10 10 10 93 96 97 91 88 95 10 94 93
sulphone (4. (1. (2. 1 1 3 (4. (3. (0. (3. (1. (0. 3 (2. (2.
3) 6) 2) (5. (3. (0. 6) 6) 9) 8) 8) 7) (6. 5) 6)
(12 (3. / 1) 0) 7) (7. (5. / (4. (5. / 4) (3. /
.6) 8) (7. (5. / 4) 6) 5) 1) (8. 2)
3) 4) 8)
Note: * inter-day repeatability values obtained by analyzing spiked samples at 2 and 10 µg kg-1
analyzed in five days

A mixture of PSA, C18 and carbon nanotubes was used as modified QuEChERS
adsorbents

Development and validation of an UPLC Q-Exactive Orbitrap MS method

LOQs of 2 µg kg-1 fulfilled the requirement of MRLs formulated by European
Union

The rate of simultaneous detection of fipronil and three metabolites in positive
samples was 51.4%