Food Chemistry: X 19 (2023) 100814

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Food Chemistry: X
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Determination of 73 multi-class pesticides in okra (Abelmoschus esculentus

L.) fruits using LC–MS/MS and GC–MS/MS and estimation of analytical
uncertainty of measurement
M.S. Pallavi a, d, R. Harischandra Naik a, b, *, K. Pavankumar a, Ratnamma a, Nandini a,
A. Shwetha a, P. Naveenkumar a, M. Paramasivam c, R. Udaykumar Nidoni a, A. Prabhuraj a,
M. Bheemanna a
a
University of Agricultural Sciences, Raichur 584 104, India
b
University of Horticultural Sciences, Bagalkot, College of Horticulture, Bangalore 560 065, Karnataka, India
c
Tamil Nadu Agricultural University, Coimbatore 641 003, India
d
KSN University of Agricultural and Horticultural Sciences, Shivamogga 577 412, India

A R T I C L E I N F O A B S T R A C T

Keywords: This study developed a method to simultaneously determine 73 multi-class pesticides in okra fruit using LC-MS/
Okra fruits MS and GC–MS/MS. The sample was extracted with acetonitrile and subsequent clean-up through dispersive-SPE
Pesticides method. The quantification level of the technique was 0.01 µg g− 1 and compliance to the MRLs fixed by the
QuEChERS
regulatory bodies like EU and FSSAI. The recovery at 10, 50, and 100 µg kg− 1 spiked levels; intra and inter-day
LC-MS/MS
GC–MS/MS
precision at 50 µg kg− 1 were found within 70–120% with RSD less than 15% with LC-MS/MS and GC–MS/MS.
Risk assessment Measurement uncertainty was in the range of 1.81 to 12.91 µg kg− 1 estimated at 50 µg kg− 1. The matrix effects
Uncertainty of measurement were slightly higher for LC than GC-compatible pesticides. Risk assessment for pesticides detected in the field and
market samples found no hazardous to the consumers except profenofos. The proposed method is highly sen­
sitive, reproducible for the complex matrix like okra, and meets the regulatory standards.

1. Introduction leading to pesticide poisoning (Darko & Akoto, 2008). Lack of awareness
of toxic effects, indiscriminate use leads to pesticide residues in vege­
Human diets are incomplete without vegetables. They constitute a tables. Chemical residues above the tolerance level (MRL) may
significant part of the preparation of various foods. Vegetables are a contribute the potential health hazards on chronic exposure and threat
good source of essential nutrients required for balanced diets viz., vita­ to food safety (Tang et al., 2018; Radwan et al., 2015; Hingmire, Oulkar,
mins (A, B1, B6, B9, C, and E), carbohydrates, proteins, antioxidants Utture, Shabeer, & Banerjee, 2015). Thus, pesticide residues quantifi­
(sulphoraphane, nasunin, allicin, and diosgenin), and minerals (Klein, cation in various foods, either raw or processed, is a critical international
1987; Naik Rathod et al., 2021). A vegetable diet reduces the risk of trade requirement for consumers (Hingmire et al., 2015).
several diseases and treats different diseases (Slavin & Lloyd, 2012). Among the vegetables, okra, Abelmoschus esculentus (L.) is a high
India is the leading vegetable producing country with an area of 10,259 export potential vegetable grown in India. India is a leading producer of
(000 ha) and production of 1, 84,394 (000 tones), and productivity of okra fruits with a total production of 6.47 million tons (3.9%) from 5.28
17.70 tones/ha (Indian Horticulture Database, 2018). Vegetable crops million hectares of the cultivated area (5.7%) with productivity of 11.63
use about 12–13% of total pesticides in agriculture, and most of them are metric tons per hectare (Indian Horticulture Database, 2018). It is
synthetic pesticides. Pesticide usage is a common practice in vegetable valued for its green fruits, rich in proteins, calcium, phosphorus, iron,
production due to visible knockdown effects on insect pests resulting in carotene, and vitamins. About 72 insect pests are recorded in the okra
toxic residues in final produce (Kumari & John, 2005). The pesticide crop from germination to harvest. Among pests, leafhopper, whitefly,
residues in agricultural produce, water, and environment samples shoot, and fruit borers were collectively causing 36–90% of yield loss

* Corresponding author at: University of Horticultural Sciences, Bagalkot, India; University of Agricultural Sciences Raichur, India.
E-mail address: harientomology@gmail.com (R. Harischandra Naik).

https://doi.org/10.1016/j.fochx.2023.100814
Received 23 February 2023; Received in revised form 12 July 2023; Accepted 26 July 2023
Available online 22 August 2023
2590-1575/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M.S. Pallavi et al. Food Chemistry: X 19 (2023) 100814

(Pal et al., 2016; Gupta et al., 2009). To avoid losses, synthetic chemical Sartorius® and sensitivity range-6 digits), centrifuge tube (Tarsons®),
pesticides are the first choice for farmers due to their high biological homogenizer (IKA®-T18), low and high volume centrifuges (Gyrozen®
efficacy and immediate effect. Farmer’s primarily relies on new gener­ − 1736R and 2236R), vortexes (REMI®), turbovap-LV evaporator (Tur­
ation chemicals like neonicotinoids (Gurbuz et al., 2014), diamide boVap®), horizontal shaker (Rotek®) were employed for the extraction
insecticide (Paramasivam & Bhuvaneswari, 2021), and pyrethroid in­ of pesticide residues from different samples used in this study.
secticides are very common and frequently applied since planting veg­
etables and are of great concern for environmental safety (Nafees & Jan, 2.3. Standard solutions
2009; Bhandari, Atreya, Yang, Fan, & Geissen, 2018). Tender fruits of
okra are harvested every 2–3 days with non-compliance to pre-harvest Stock solutions of each pesticide compound were weighed (10 ± 0.1
intervals, and due to its short fruits plucking periods, pesticide residue mg) using a calibrated analytical balance (7 digit microbalance: Sarto­
may likely occur (Paramasivam & Bhuvaneswari, 2021). A great ne­ rius®) in a volumetric (10 mL) flask and dissolved with 10 mL methanol
cessity of screening the vegetable foods for pesticides before being for LC and ethyl acetate for GC compatible. Further, the final concen­
subjected to export demands high throughput techniques to develop the tration in the primary stock solution was calculated using the formula:
analytical methods.
( ) Weight of CRM (mg) × Purity of CRM (%) × 1000
Pesticide residues in the harvested vegetable produce beyond their Concentration μg mL− 1 =
tolerance limits (MRL) are the trade barriers at the export point and non- Final volume (mL) × 100
compliance to the food safety regulations (FSSAI, 2018; European
Commission, 2008). MRLs are very low for most of the pesticides All the solutions were prepared in different volumes calibrated class
required an efficient method to meet global food standards. A variety of ‘A’ glassware. An intermediate stock solution of 100 µg mL− 1 was pre­
analytical methods were reported for the simultaneous quantitative pared, drawing 1 mL of standard stock solution (1000 µg mL− 1) into a
analysis of pesticides in fruits and vegetables viz., GC with ion trap 10 mL volumetric flask and volume made-up. Working standard mixture
(Savant et al., 2010); GC-ToF MS and LC-MS/MS (Banerjee et al., 2008 (10 µg mL− 1) was prepared by mixing a known amount of individual
and 2012; Walorczyk, 2008), LC-APCI-MS/MS for multi-class pesticides solution through serial dilution techniques. All these solutions were
in rice (Caldas et al., 2011); LC-MS/MS in spices (Yogendrarajah, Van, stored in a refrigerator at − 20 ◦ C. The calibration standard solution
De Meulenaer, & De Saeger, 2013); LC-MS/MS & GC–MS/MS for mul­ ranging from 0.01 to 1.0 µg mL− 1 was prepared and injected to LC-MS/
tiresidue in pigeonpea (Naik et al., 2021) and rice (Harischandra et al., MS and GC–MS/MS. The matrix match standards having similar linear
2021) employed for rapid detection and quantification of residues. For concentrations were prepared simultaneously using untreated okra fruit.
an analytical method, it is very essential to have the uncertainties
associated with it that greatly influence the analytical results for making 2.4. Sample preparation
a decision to the real samples. Most of the previous work has lack of
measurement uncertainties. For a rapid detection of pesticide residues in Approximately 2 kg okra fruits (pesticide-free) were chopped, ho­
vegetable foods is always challenging due to the complex matrix nature. mogenized (Robo-Coup), and collected in a cleaned inert container. A
This is achieved following suitable methods, and modifications provide test portion (10 g) of the sample was transferred into 50 mL centrifuge
solutions to practical applications to the complex matrices. In this study, tubes, and acetonitrile (20 mL) was added and homogenized using a
quick, sensitive, and reproducible analytical methods using LC-MS/MS homogenizer for 3 min. Then 1.5g NaCl was added and vigorously
and GC–MS/MS were developed and validated in okra fruits following shaken 30 sec and centrifuged the content at 13416xg for 10 min. The
European commission validation guidelines. The technique was applied upper organic layer (10 mL) was carefully transferred into a test tube
to real sample analysis, estimated the associated uncertainty, and containing the 5g anhydrous Na2SO4 to remove the moisture. For d-SPE
assessed the risk of consuming vegetables detected with pesticides. clean-up, 8 mL of extract was transferred into a centrifuge tube con­
taining 200 mg PSA and 1.0 g anhydrous MgSO4 added. The tubes were
2. Material and methods shaken well and centrifuged 13416xg for 5 min to get a clear solution.
The supernatant (1 mL) solution was filtered through a 0.2 μm syringe
2.1. Pesticide selection filter and directly injected (2 μL) into LC-MS/MS. In GC–MS/MS anal­
ysis, 2 mL of the clear supernatant was concentrated using a nitrogen
Seventy-three pesticides were selected in this study, which included flash evaporator, reconstituted with 1.0 mL ethyl acetate, and filtered
both GC and LC suitable molecules. Certified reference materials (CRM) into GC vials using a 0.22 µm PTFE nylon filter, and 1 μL was injected
with known purity (≥98.0%) having traceability to ISO/IEC 17,034 into GC–MS/MS.
were purchased from Dr. Ehrenstorfer (Augsburg, Germany). The names
of these pesticides are presented in Table S1. 2.5. LC-MS/MS parameters

2.2. Chemical, reagents and apparatus LC-MS/MS (LC-MS 8040, Shimadzu®) system attached with UHPLC
for separation and triple quadrupole detector was used to confirm and
The solvents (>99.9% purity) viz., methanol, ethyl acetate, and quantify multi-class pesticides in okra fruits. The instrument was
acetonitrile (LC-MS grade) were procured from Merck (Mumbai, India). controlled with LabSolution® software, version 1.5. Chromatographic
Anhydrous sodium chloride, magnesium sulfate, sodium acetate, and separation of pesticides (39) done with C18 column (Octadecylsilyl; 2
sodium sulfate (>99% purity) were purchased from Himedia (Banga­ mm i.d × 150 mm, 2.2 µm particle size). Binary gradient program with
lore, India). Ammonium formate and formic acid (90% purity) were mobile phase (A) consisted of 5 mM ammonium formate, 2 mL meth­
obtained from Empart (Hyderabad, India). Primary secondary amine anol, and 0.01% formic acid and made up to the volume of 100 mL using
(PSA, 40 µm size) was purchased as dispersive-solid phase extraction HPLC water (2:98, v/v methanol: water) and (B) consisted of 5 mM
sorbent from Agilent make (Agilent Technologies India Pvt. Ltd., Ban­ ammonium formate, 0.01% formic acid made up to the volume of 100
galore, India). Ultrapure water with a resistivity of 18.2 MΩ was mL with 100% methanol. Flow rate and injection volume of 0.4 mL
collected from a Milli-Q (Merck Millipore, USA) water purification min− 1 and 2 µL, respectively was maintained throughout the study. The
system installed in the laboratory. mobile phase gradients program for the binary pump was initially 60% A
GC–MS/MS (Shimadzu, GCMS TQ 8030®) and LC-MS/MS (Shi­ and 40% B for 15 min. followed by 100% B for five min. and then 60% A
madzu, LC-MS 8040®) were used for method development, qualitative and 40% B for 5 min. The column temperature was maintained at 40 ◦ C.
and quantitative analysis of pesticides. Analytical balance (Make- The cycle time and total time program were 0.234 sec and 19 min.,

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M.S. Pallavi et al. Food Chemistry: X 19 (2023) 100814

respectively. The mass spectrometer was operated in electrospray ioni­ 2.7.2. Matrix effect (ME)
zation (ESI) with both positive and negative modes. At the same time, The complex matrices were responsible for suppressing and
maximum compounds were separated through ESI positive mode. The enhancing analyte signals or responses in LC-MS/MS and GC–MS/MS. It
triple quadrupole mass was employed for analyte fragmentation with was evaluated using the following formula.
optimized voltage and collision energy to get the highest sensitivity. The
Matrix effect (%) = (bm-bs)/bs × 100,
specific MS/MS parameters such as interface current (0.1 µÅ), heat block
temperature (400 ◦ C), and desolvation line temperature (250 ◦ C) were where bm and bs are the angular coefficient from the matrix and the
optimized and acquired the MS data. High pure nitrogen (99.99% pu­ solvent linearity studies, respectively (Silva, Habermann, Marchi, &
rity) was used as nebulizer and drying gas at 2.9 and 15 L min− 1, Zocolo, 2012; Naik et al., 2021).
respectively. High pure argon gas (99.99% purity) 230 kpa was used for
collision energy diffraction (CID). Mass scan speed (6000 usec-1) and 2.7.3. Limit of quantification
dwell time 1 msec were set. The LOQ is the lowest spiked concentration that could be quantified
and recorded acceptable recovery in the range of 70–120% and relative
standard deviation of ≤ 20%. Further, the lowest concentration level for
2.6. GC–MS/MS instrumentation the instruments such as LC-MS/MS and GC–MS/MS could quantify the
residue less than MRLs of regulatory bodies.
GC–MS/MS (TQ-8039, Shimadzu®, Kyoto, Japan) was used to
separate and quantify 34 pesticides in okra fruits. The gas chromato­ 2.7.4. Trueness (Recovery)
graph is equipped with MS triple quadrupole coupled with an AOC-20i The trueness or recovery was conducted spiking pesticide standard
injector and AOC-20 s auto-sampler. The GC–MS/MS is controlled using mixture at 10, 50, and 100 μg kg− 1 to the blank okra samples and
Labsolution® software (Version 1.5). The separations of target pesti­ extracted following the procedure standardized. Matrix matched cali­
cides were performed using a capillary fused silica HP 5 MS column (30 bration standards responses were used to compare the responses recor­
m × 0.25 mm i.d., 0.25 µm film thickness) with splitless injection (2 µL) ded in the spiked samples and quantify the residues and calculated
mode. The oven temperature program was as follows; the initial tem­ percent recovery.
perature was set at 60 ◦ C for 2 min, ramped at the rate of 25 ◦ C min− 1 to
150 ◦ C, 3 ◦ C min− 1 rate to 200 ◦ C, 8 ◦ C min− 1 to 280 ◦ C for 5 min. and 2.7.5. Precision (intra and inter-day)
finally ramped to 300 ◦ C at 25 ◦ C min− 1 for 3 min. All the pesticides Intraday and interday precision (Repeatability-RSDr and
clearly separated within the total run time of 25 min. The carrier gas was Reproducibility-RSDwr) was assessed separately at 50 µg kg− 1 spiking
helium (99.999% purity), with a constant flow rate of 1.5 mL min− 1, level. Intraday precision was performed in six blank extract samples
surge pressure of 250 kpa, and injector temperature 280 ◦ C was using the standardized method and recorded the LC-MS/MS and
maintained. GC–MS/MS response. Similarly, the interday precision test was con­
MS parameters were as follows; transfer line and ion source tem­ ducted on two different days and calculated the recovery and RSD.
perature was set at 280 and 250 ◦ C, respectively. Ionization was done
through positive electron impact (EI) at –70 eV. The MRM optimization 2.7.6. Ion ratio
was performed by obtaining the product ion scan and collision energy For MS/MS triple quadrupole, a minimum of 2 product ions is
for each transition. The solvent delay was fixed at 3 min. Argon (Ar) and essential as per the SANTE guidelines. This method selected two product
helium (He) with a flow rate of 1.50 and 2.25 mL min− 1 were main­ ions and a precursor ion for each pesticide compound after injecting
tained as collision and quench gases. The mass range was between 50 individual pesticides to LC-MS/MS and GC–MS/MS. The ion ratio was
and 550 m/z. calculated based on the peak area or intensity ratio of low intense ions to
that high intense ions. The ion ratio value was calculated as,

2.7. Method validation Ion ratio =

Peak area (confirmation ion)
× 100
Peak area (quantitation ion)
The European Commission document SANTE/12682/2021 was fol­ The ion ratio from sample extracts was estimated, and ions recorded
lowed to validate the developed method with LC-MS/MS and GC–MS/ less than or equal to ± 30% (relative) were considered for confirmation
MS. The criteria viz., linearity, matrix effect, limit of quantification of the analytes.
(LOQ), trueness (recovery), precision (intraday), precision (interday),
ion ratio, and retention time were assessed for 73 pesticides in the okra 2.7.7. Retention time (RT)
matrix. The good retention time of target analytes in the LC-MS/MS and
GC–MS/MS chromatogram was examined from the calibration standard
2.7.1. Sensitivity/linearity and matrix match standard solutions with a tolerance limit of ± 0.1 min.
A linearity study was performed, preparing different concentrations
of pesticide mixture ranging from 0.01 to 1.0 μg mL− 1 in solvent stan­
dard and matrix extract of okra fruit. Estimated the response (residuals) 2.8. Estimation of measurement uncertainty
of linear concentration after injection into the instruments based on the
linear regression equation. The linearity graphs were drawn by plotting Uncertainty due to repeatability test from the intra-laboratory vali­
concentrations against the response and recording the coefficient of dation process (also called type ’A’ uncertainty: U), combined uncer­
determination (R2). Residuals were calculated using the following tainty (Uc), and expanded uncertainty (Uexp) was estimated for all the
formula: insecticides following EURACHEM/CITAC Guide (Ellison & Williams,
2012; NABL 164, 2016; Naik et al., 2021; Harischandra et al., 2021). The
Deviation of back-calculated concentration (%) = (Cmeasured – Ctrue) × 100/ mathematical formulae for estimation of uncertainty (Uexp at 95%
Ctrue confidence limit) are given below.
( ) Area of sample − Intercept (c)of linearity Uncertainty due to repeatability
Measured concentration ng g− 1 =
Slope (m)of linearity Standard Uncertainty
U=
− 1 Mean
Ctrue– Linearity standard concentration (ngg ).

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M.S. Pallavi et al. Food Chemistry: X 19 (2023) 100814

Combined Uncertainty (Type ’B’ Uncertainty), standardized protocol and quantified the residues. Further, the highest
residues for detected pesticides were considered to assess the risk of
UC = Mean value of repeatability result
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ following the Hazard Index (HI) model. Initially, the estimated daily
× summation of square of all individual uncertainty from U1 to U11 intake (EDI) was calculated by multiplying the residues of individual
pesticides with per capita okra fruits (vegetables as whole) consumption
Coverage factor (k) at 95 % Confidence level
rate (kg day− 1) by the adult and children. Then, the hazard index was
(UC )4 estimated by dividing EADI of individual pesticides through their cor­
Veffective = (3) responding ADI (Acceptable Daily Intake). The safety of the okra fruits
(U1 )4 (U2 )4 4
+ + ⋅⋅⋅ + (U n)
(d.f ) (d.f ) (d.f )
collected from the market and fields was categorized based on hazard
Where, UC is the Combined Uncertainty, U1, U2, …Un are relative index values. It is presumed that if HI is>1, then vegetable food is not
uncertainty of individual component and d.f is degrees of freedom for safe for consumption (Darko & Akoto, 2008; Kumari and Jhon, 2019).
each component.
Expanded Uncertainty (at 95% confidence; k = 2) is estimated as 3. Results

Uexp = Uc × k; (4) 3.1. Optimization of LC-MS/MS parameters

Where, Uexp is Expanded Uncertainty; k is the coverage factor (k) at
The optimized liquid chromatographic conditions with different
95% Confidence level and UC is the Combined Uncertainty.
mobile phases (A and B) utilized to separate the pesticides provided
proper ionization. The stationary phase of the C18 column (octade­
2.9. Quality assurance cylsilyl, 2 mm i.d × 150 mm × 0.2 µm) separated and recorded good
peak shape for all the 39 pesticides with a gradient LC time program of
Different sources of uncertainty were taken into account, such as 25 min in LC-MS/MS (Fig. 1). The most abundant m/z ions (mass-to-
Uncertainty due to repeatability (6 replicate) (U1), balance for weighing charge) for each pesticide were recorded through full scan mass spec­
of the sample (U2) and weighing of standards (U3), 10 mL volumetric trums by injecting 2 µL of individual pesticide standards. The protonated
flask for preparation of 1000 µg mL− 1 stock solution (U4), 10 mL molecular ion (M + H)+ was determined and recorded as precursor ion
volumetric flask for preparation of 100 µg mL− 1 intermediate solution (Table S1). Further, 2 µL of 0.1 µg mL− 1 pesticide standard mixture was
(U5), 10 mL volumetric flask for preparation of 10 µg mL− 1 working injected into the LC-MS/MS to optimize the MRM (multiple reaction
solution (U6), 10 mL volumetric flask for preparation of 0.5 µg mL− 1 monitoring) transitions and acquisition for a higher abundance of the
working solution (U7), 100 µL micropipette for preparation of calibra­ fragmented ions through positive electrospray ionization (ESI + ).
tion curve (U8), calibration curve (U9), recovery of particular analyte Finally, dissociation with argon gas was induced, and the different
(U10) and certified reference material (U11) were calculated. Further, collision energy was tested to record the most abundant productions.
the combined uncertainty (Uc) was calculated by multiplying the mean Three different ions viz., a precursor, and two product ions used to
value of repeatability result and summation of square of all individual quantify and confirm the target pesticide in the samples. The selected
uncertainty from U1 to U11. Coverage factor, k = 2 at 95 % confidence MRM transitions recorded higher sensitivity and selectivity for the
level, was considered for estimation of expanded uncertainty (Uexp). pesticides at 0.01 to 1.00 mg kg− 1 in the okra fruit matrix (Table S1).

2.10. Screening of unknown samples and risk assessment 3.2. Optimization of GC–MS/MS

Twenty-five okra fruit samples from the field and market were The targets of 34 pesticides were determined in a single GC run with
collected and screened for the pesticide residues following validated trifluralin compound was first detected with the retention time of 12.34
methods. The samples were extracted and clean-up with the min, and last deltamethrin compound was detected with the retention

1.
Fig. 1. MRM Chromatogram of 39 LC pesticides recovery in okra matrix fortified at 0.01 mgkg−

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M.S. Pallavi et al. Food Chemistry: X 19 (2023) 100814

time of 34.64 min (Fig. 2). All the target pesticides were eluted with range of 70.69 to 106.36%, with RSD less than 12.86%. The precision
good resolution and a retention time deviation of ± 0.1 min. The with inter-day test recorded the recovery of 73.31 to 104.23 % with RSD
GC–MS/MS parameters were optimized to detect, confirm, and quantify less than 15% for pesticide tested in LC-MS/MS. The intraday precision
the residue of selected pesticide residues in okra fruit. The mass spectra test recorded 72.31 to 117.86 % recovery, and inter-day precision
obtained from the full scan or total ion chromatogram of target com­ recorded 74.14 to 114.97% recovery and RSD less than 15% for the
pounds showed the most abundant ion at m/z (100% relative abun­ GC–MS/MS. Overall, the present study on method validation with okra
dance) was selected as precursor ion and productions selected for fruit matrix fulfilled the SANTE/12682/2019 guidelines. The ratio be­
confirmation of the pesticides in the okra fruit matrix. Further, product tween quantifier and qualifier ions was used as a confirmatory tool for
ions scan was performed with different Collision Energies (CE) to in­ the pesticides in the okra fruit matrix. The ion ratio of both LC-MS/MS
crease the sensitivity of target compounds. For each pesticide, multiple and GC–MS/MS methods was less than 30%. With the chromato­
reaction monitoring (MRM) transitions with appropriate CEs were graphic conditions set in the present methods, selected pesticides were
determined (Table S1) and further simplified by using the smart MRM separated, and retention time of all pesticides on okra fruit in GC–MS/
optimization tool (SANTE/12682/2019). MS and LC-MS/MS were found to be within the acceptable limit of ± 0.1
min.
3.3. Method validation
3.4. Matrix effect
The method optimized 73 pesticide residues in okra fruit using LC-
MS/MS and GC–MS/MS (Table 1 & 2). All the pesticides were deter­ Matrix effect is common in different food matrices, and matrix match
mined in a single run time of 25 and 41.07 min in LC-MS/MS and calibration standards are routinely used to nullify the matrix effect in the
GC–MS/MS, respectively, with the coefficients of determination (R2 > chromatographic method. It was assessed using the blank extracts
0.998) with the linear concentration of 0.01 to 1.00 mg kg− 1. The LOQ (without target pesticides) of okra fruit for matrix match calibration.
of all the pesticides in okra fruits was considered 10 µg kg− 1 for both GC Most of the selected pesticides showed an acceptable matrix effect
and LC techniques due to the acceptable recovery recorded with this within the range of − 2.44 to 37.24 %. The matrix effect observed in the
proposed method. This level is less than MRLs (EU and FSSAI) of pes­ present method for okra fruit sample was considered to be less to
ticides considered in this study. moderate matrix effects as per Ferrer, Lozano, Agüera, Jiménez, and
The analytical method was found to be efficient for the okra fruit. Fernández (2011), who classified the matrix effects into three different
The obtained recoveries were ranged between 70 and 120%. Okra fruit categories viz., less (<20%), moderate (20–50%) and strong (>50%)
was spiked at 10, 50, and 100 µg kg− 1 level recorded recovery in the based on signal suppression and or enhancement. The calculated matrix
range of 72.23 to 116.83%, 71.94 to 109.06%, and 73.94 to 116.57%, effect was much below the maximum threshold limit of 20% signal
respectively, and RSD at different fortification levels were found less suppression or enhancement. For more realistic estimation and quanti­
than 15% for LC-MS/MS (Table 1 & Fig. 2). For GC compatible pesti­ fication of pesticide residues in the samples, the response of matrix-
cides, recovery was in the range of 72.14 to 119.70 %, 70.03 to 88.91%, matched calibration standard is suggested to compare with pesticide
and 75.07 to 115.72% with relative standard deviation less than 15% at response in the unknown samples, and quantified pesticide concentra­
10, 50, and 100 µg kg− 1 levels, respectively (Table 2 & Fig. 5). Similar tion would be the actual value (Silva et al., 2012; Naik et al., 2021 &
recoveries (<90%) were observed for okra at 25 μg kg− 1 (Banerjee et al., Harischandra et al., 2021). Overall, the matrix effect with both tech­
2012). The recovery in both techniques was found acceptable as per the niques was significant. Comparing the matrix effects observed in grape,
validation guidelines. mango, capsicum, okra, and drumstick for multi-class pesticides was
Intraday precision estimation at 50 µg kg− 1 recorded the recovery moderate to low matrix effect (Jadhav et al., 2015). The present study

Fig. 2. GC–MS/MS MRM chromatogram for standard concentration at 10 µgkg− 1.

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M.S. Pallavi et al. Food Chemistry: X 19 (2023) 100814

Table 1
Recovery, precision (intra and inter day), matrix effect, EU- MRL and uncertainty for pesticides in okra fruits using LC-MS/MS.
Pesticide Recovery % (RSD %) Intra-day precision (50 Inter-day precision (50 Matrix effect EU MRL (mg/ Uc at 50
µg kg− 1) µg kg− 1) (%) kg) µgkg− 1
1 1 1
10 µg kg− 50 µg kg− 100 µg kg− Recovery RSD Recovery RSD
(%) (%) (%) (%)

Thiacloprid 101.7 (8.78) 104.50 101.24 98.67 9.29 102.23 10.09 8.19 0.01 12.48
(10.48) (8.30)
Buprofezin 88.55 95.80 (9.17) 101.55 93.67 9.10 94.36 12.63 12.74 0.01 8.51
(10.32) (8.66)
Metalachlor 98.80 (8.12) 109.06 98.62 96.68 9.61 92.31 14.89 10.79 0.05 10.50
(10.53) (10.50)
Imidacloprid 96.21 97.36 (8.69) 92.30 (9.91) 92.38 11.05 98.31 6.41 23.19 0.50 12.48
(10.35)
Dimethoate 101.41 94.89 (7.34) 110.96 96.36 9.98 95.26 13.28 27.24 0.01 9.97
(7.27) (8.60)
Coumatetryl 81.90 (8.26) 89.18 (8.72) 82.11 84.28 9.90 85.21 5.58 24.14 – 9.97
(10.30)
Triadimenol 73.05 78.98 77.19 (9.77) 76.36 10.39 76.16 6.09 16.53 0.01 10.21
(11.94) (10.39)
Triadimefon 99.63(8.49) 100.29 116.57 99.86 9.87 98.31 5.30 32.43 0.01 11.62
(9.18) (10.35)
Thiobencarb 86.35 82.14 82.56 (9.60) 86.36 12.86 87.31 4.31 − 3.59 0.01 11.63
(13.36) (11.69)
Spinosad 111.56 93.19 (6.13) 80.52 94.35 10.10 96.28 5.51 1.96 0.02 12.57
(9.79) (10.03)
Phosalone 112.05 110.59 102.99 106.36 12.54 104.21 12.51 7.52 0.01 10.25
(7.52) (8.52) (10.02)
Methoxyfenozide 74.37 71.94 (7.82) 73.94 (9.82) 70.69 9.98 73.31 10.33 15.90 0.01 9.80
(11.03)
Hexythiazox 102.78 104.49 102.29 100.26 12.50 104.23 13.57 18.24 0.50 11.99
(13.57) (9.26) (9.71)
Fenpyroximate 88.54 92.02 92.73 (9.48) 90.19 11.24 93.21 11.22 15.78 0.01 12.05
(11.63) (10.65)
Carbendazim 104.42 93.30 (9.25) 95.53 (9.01) 94.86 11.05 95.07 9.18 28.10 2.00 11.29
(9.56)
Carbaryl 89.53(8.42) 90.72 (8.57) 101.61 91.23 10.59 94.26 6.63 10.74 0.01 9.07
(8.41)
Triazophos 95.68(7.54) 84.15 90.83 83.18 10.53 86.26 14.55 19.80 0.01 10.87
(10.57) (10.26)
Carbofuron 77.72 (8.98) 82.28 (7.51) 85.08 (9.52) 86.68 9.31 85.21 10.02 12.07 0.002 8.98
Bitertanol 103.76 100.03 98.90 (8.26) 97.68 10.86 96.28 9.57 14.46 0.01 12.26
(8.80) (9.67)
Bendiocarb 89.51(6.52) 87.04 91.09 (8.05) 88.25 12.08 84.23 12.85 20.37 – 10.04
(10.55)
Benalaxyl 116.81 94.69 (9.04) 92.56 (8.94) 94.36 11.88 98.23 12.25 13.63 0.05 9.68
(9.39)
Acephate 76.63(6.06) 83.54 84.81 (9.54) 87.86 12.19 89.26 5.86 27.21 0.01 10.71
(10.19)
Pymetrozine 81.69 81.23 87.10 (9.33) 84.29 10.66 88.13 7.99 9.80 0.01 11.66
(12.22) (12.25)
Omethoate 88.92(9.84) 82.94 86.15 (9.94) 81.69 12.65 83.23 10.30 10.34 0.01 12.84
(10.01)
Metribuzin 73.87 78.83 84.32 (9.84) 74.39 12.60 81.06 12.92 21.56 0.1 9.97
(11.32) (12.50)
Metalaxyl 109.25 100.38 101.27 101.29 10.42 103.26 9.38 − 4.38 0.01 9.57
(9.43) (8.76) (9.72)
Emamectin 113.69 103. 61 99.47 (8.03) 100.17 11.05 99.86 8.10 4.93 0.02 8.35
Benzoate (6.65) (9.69)
Tetraconazole 106.24 90.06 (7.12) 92.78 (9.23) 94.26 10.20 96.16 13.13 − 11.05 0.02 9.65
(6.18)
Quinalphos 91.61(8.55) 101.40 101.79 102.30 9.95 100.39 8.80 20.97 0.01 12.19
(1132) (8.83)
Profenofos 72.23 78.77 (8.09) 83.26 (9.82) 74.26 11.36 74.19 8.32 25.58 0.01 12.30
(14.53)
Phosphomidan 99.18 106.13 95.38 102.36 12.62 104.23 12.68 − 9.04 0.01 9.87
(11.32) (9.79) (10.36)
Pendimethalin 95.60 100.06 102.77 96.29 12.38 98.38 9.48 13.51 0.05 10.03
(12.01) (11.42) (9.41)
Difenconazole 96.76 (9.30) 97.56 101.44 99.16 8.90 100.01 10.68 − 6.93 0.06 9.96
(10.35) (10.21)
Pretilachlor 106.81 95.17 (8.65) 95.77 (9.50) 102.36 10.42 97.62 12.18 20.00 – 10.96
(8.88)
Paclobutrazole 105.30 91.98 99.22 (8.77) 92.38 9.45 90.26 14.99 11.51 0.01 12.91
(9.68) (10.74)
Chlorantraniliprole 111.91 100.04 96.89 103.68 12.83 103.00 5.44 2.54 0.60 11.63
(7.06) (11.69) (10.43)
(continued on next page)

6
M.S. Pallavi et al. Food Chemistry: X 19 (2023) 100814

Table 1 (continued )
Pesticide Recovery % (RSD %) Intra-day precision (50 Inter-day precision (50 Matrix effect EU MRL (mg/ Uc at 50
µg kg− 1) µg kg− 1) (%) kg) µgkg− 1
1 1 1
10 µg kg− 50 µg kg− 100 µg kg− Recovery RSD Recovery RSD
(%) (%) (%) (%)

Isoproturon 104.83 93.20 (9.76) 97.95 94.28 12.21 96.68 8.82 15.59 0.01 10.50
(7.99) (10.37)
Hexaconazole 84.23 (7.87) 88.59 81.10 (8.71) 86.35 9.89 87.23 11.25 23.84 0.01 8.40
(11.52)
Penconazole 98.51 105.09 102.40 101.34 9.82 96.25 14.81 8.00 0.01 10.05
(12.38) (8.36) (9.56)

MRL-maximum residue limit; EU-European Union; Uc-Combined Uncertainty.

Table 2
Recovery, precision (intra and inter day), matrix effect, EU-MRL and uncertainty for pesticides in okra fruits using GC–MS/MS.
1 1
Pesticide Recovery % (RSD %) Recovery (RSD %) @ 50 µgkg− Matrix effect (%) EU MRL (mg/kg) Uc at 50 µgkg−
1 1 1
10 µgkg− 50 µgkg− 100 µgkg− Intra-day Inter-day

Fluchloralin 94.77 (14.87) 77.10 (12.07) 76.06 (5.22) 91.03 (14.92) 91.02 (17.78) 12.65 – 2.69
alpha-BHC 79.53 (14.48) 79.15 (10.66) 77.83 (8.79) 78.52 (10.57) 80.70 (10.03) 6.71 0.01 3.38
beta-BHC 89.51 (13.53) 72.95 (9.10) 83.17 (7.80) 88.57 (14.99) 86.75 (14.06) 6.40 0.01 2.43
Diazinon 113.15 (9.43) 70.78 (14.12) 79.78 (13.05) 93.57 (16.66) 97.33 (16.12) 6.86 0.01 3.32
Trifluralin 89.83 (13.39) 79.06 (12.14) 78.03 (7.27) 90.01 (15.89) 91.87 (11.72) 20.58 0.01 10.70
Tri-allate 102.06 (12.70) 77.88 (10.98) 75.95 (12.10) 84.12 (16.88) 84.90 (16.57) 6.80 0.1 6.38
Iprobenfos 108.10 (9.76) 83.24 (10.02) 98.38 (8.30) 106.59 (11.74) 111.29 (10.01) 15.57 – 3.97
Propanil 117.76 (11.26) 78.32 (11.41) 90.11 (6.67) 117.86 (11.79) 105.73 (12.11) 5.00 0.01 7.67
Chlorpyrifos-methyl 115.57 (15.26) 73.39 (14.29) 82.51 (6.26) 83.64 (17.59) 92.08 (17.59) 12.73 0.01 2.28
Parathion-methyl 76.39 (15.51) 74.88 (7.58) 91.51 (7.47) 96.60 (12.34) 93.17 (9.98) 14.01 0.01 3.58
Alachlor 118.00 (9.14 83.76 (9.71) 97.66 (8.66) 106.00 (10.83) 110.44 (9.91) 8.96 0.01 1.81
Heptachlor 91.98 (12.65) 77.14 (14.3)1 75.07 (12.07) 73.46 (15.16) 75.54 (16.82) 13.88 0.01 1.93
Fenitrothion 116.34 (4.58) 70.77 (10.22) 87.10 (7.42) 95.27 (8.79) 102.22 (16.74) 14.29 0.01 6.08
Aldrin 98.58 (10.79) 71.97 (9.54) 76.58 (8.63) 74.69 (15.72) 72.31 (14.66) 4.12 0.02 9.02
Chlorpyrifos 112.04 (11.51) 88.91 (15.59) 79.27 (5.35) 80.50 (16.13) 87.28 (16.28) 6.40 0.01 2.38
Parathion 75.36 (12.30) 76.66 (7.38) 82.68 (6.07) 91.81 (13.96) 96.23 (9.01) 5.92 0.05 3.18
Chlorofenvinphos 117.22 (8.59) 74.88 (12.68) 92.13 (8.34) 97.53 (15.97) 107.82 (14.11) 37.24 0.01 6.36
Phenthoate 83.23 (10.71) 84.47 (7.89) 86.68 (8.42) 108.55 (16.23) 110.96 (13.17) 13.54 – 1.96
Butachlor 93.81 (11.25) 74.47 (14.85) 99.44 (9.66) 95.81 (11.09) 101.10 (10.05) 19.20 – 3.63
p,p’-DDE 76.66 (7.31) 75.31 (9.74) 79.91 (3.66) 83.08 (11.36) 78.62 (13.53) 3.95 0.05 3.21
Endrin 79.83 (12.41) 71.77 (13.48) 84.12 (7.24) 79.76 (11.45) 86.23 (19.22) − 5.39 0.01 10.11
beta-Endosulfan 119.70 (14.82) 75.97 (13.18) 79.38 (13.06) 116.16 (30.99) 98.73 (28.26) − 8.70 0.05 11.27
o,p’-DDT 87.41 (10.73) 70.68 (8.85) 88.80 (3.38) 92.52 (13.28) 88.01 (10.71) − 3.36 0.05 4.67
Endosulfan sulfate 86.70 (11.75) 76.02 (15.15) 115.72 (14.41) 93.56 (44.20) 93.73 (29.05) 1.00 0.05 2.78
p,p’-DDT 72.14 (11.61) 84.36 (14.16) 77.40 (12.35) 77.10 (16.12) 75.29 (18.33) − 2.44 0.05 5.69
Bifenthrin 99.98 (12.14) 82.23 (6.90) 101.08 (7.26) 99.16 (10.37) 99.05 (9.80) − 18.18 0.20 6.17
Fenpropathrin 100.20 (12.45) 81.93 (9.03) 108.23 (10.14) 106.85 (11.30) 114.97 (10.07) − 9.40 0.01 11.99
lambda-Cyhalothrin 78.42 (12.30) 86.09 (5.63) 103.51 (6.87) 111.77 (8.67) 114.91 (7.29) 3.15 0.30 9.36
Permethrin 72.60 (12.70) 78.47 (6.46) 81.93 (4.00) 100.58 (7.58) 87.31 (10.22) 0.77 0.05 6.30
Cyfluthrin 79.53 (13.14) 77.95 (7.62) 80.23 (2.70) 95.62 (8.33) 93.82 (8.34) − 6.50 0.02 12.16
Cypermethrin 73.24 (15.03) 70.25 (6.02) 83.97 (3.78) 95.14 (8.17) 92.25 (7.92) − 7.96 0.50 12.39
Etofenprox 78.79 (12.10) 79.78 (14.95) 77.83 (13.45) 79.73 (14.78) 76.22 (14.09) − 6.59 0.01 12.27
Fenvalerate 77.91 (13.86) 74.65 (12.32) 79.91 (9.42) 96.94 (15.92) 72.09 (18.98) − 9.72 0.02 11.16
Deltamethrin 70.08 (14.21) 70.03 (10.20) 87.94 (5.10) 83.46 (9.66) 85.66 (7.21) − 9.58 0.01 9.54

MRL-maximum residue limit; EU-European Union; Uc-Combined Uncertainty.

recorded a similar observation with okra matrix for 73 pesticides with MRLs of that particular contaminant. In such cases, the combined un­
GC–MS/MS and LC-MS/MS. certainty is considered a tool to decide analytes’ fate, whether above or
below MRLs, and declare the results (NABL, 164). In the present study,
3.5. Estimation of measurement of uncertainty 73 multi-class pesticides recorded acceptable uncertainty measurement
values (Table 1 &2). Similar results were reported previously found in
Uncertainty due to repeatability was estimated for a minimum of 6 made tea and tea infusion and spent leaves (Kanrar, Mandal, & Bhat­
different consecutive extraction of the okra fruit samples spiked at 50 µg tacharyya, 2010; Banerjee et al., 2007); the uncertainty of measurement
kg− 1 produced acceptable recoveries and could contribute significantly for aflatoxin maize and fig (Stadler, Sulyok, Schuhmacher, Berthiller, &
to the measurement uncertainty. The uncertainty values were ranged Krska, 2018), pigeonpea and rice grain (Naik et al., 2021; Harischandra
from 1.81 to 12.91 µg kg-1estimated at 50 µg kg− 1. Combined uncer­ et al., 2021).
tainty is the inclusion of all the relative uncertainty of individual
component viz., sample weight, weighing of standards, preparation 3.6. Real samples analysis and risk assessment
stock (1000 μg mL− 1), intermediate (100 μg mL− 1) and working solution
(10 μg mL− 1 and 0.5 μg mL− 1), use of micropipette (100 μL) for linearity, A total of 25 okra fruit samples were screened separately for both LC-
calibration curve, recovery of analyte and certified reference material. MS/MS and GC–MS.MS. The fenpyroxymate, carbendazim, profenofos,
Combined uncertainty is a criterion for deciding on the sample results acephate, imidacloprid, hexaconazole, emamectin benzoate, tri­
when a quantified analyte is reported value near or equal to the LOQ and ademenol and bifenthrin was detected in the farm field and market okra

7
M.S. Pallavi et al. Food Chemistry: X 19 (2023) 100814

fruit samples analyzed by LC-MS/MS and GC–MS/MS at residue con­ quantification of multi-group pesticides.
centrations of 0.01 to 0.11 mg kg− 1 and 0.01 to 0.16 mg kg− 1, respec­ A method using LC-MS/MS was reported to determine fipronil and its
tively for field and market samples (Table 3 & 4; Fig. 3 & 6). Further, the metabolites simultaneously, and difenoconazole in okra provided
data subject to the risk assessment revealed that the Hazard Index value 80–107% recoveries with an RSD range between 4 and 17% LOQ ranged
for the maximum residues for profenofos was>1. It is having the risk to 1 to 5 ng g− 1 (Hingmire et al., 2015). However, Banerjee et al. (2012)
both the consumer category (children and adults). Whereas the in­ reported a multi-residue method for different fruits and vegetables,
secticides detected in the market samples did not have risk except the including okra using GC-EI-MS/MS with LOQ of less than 10 µg/L and
profenofos. None of the other insecticides has significant toxicological acceptable recovery range of 70–110% and RSD less than 20% is a
risk in this study, and that would not cause any hazardous to the similar method concerning proposed method in the present investiga­
consumers. tion. Whereas, Jadhav et al. (2015) reported a similar method for pes­
ticides in fruits and vegetables with 70–120% recoveries and RSD less
4. Discussion than 20% are the latest methods available. Present methods are superior
to those published previously in terms of sensitivity, accuracy, repeat­
4.1. Method comparison ability, and reproducibility. A significant part in these methods is
confirmation of the residues in the market and field collected samples.
The present investigation reported a highly sensitive and reproduc­ An analytical method needs to ensure the analyte detection in the
ible analytical method with LC-MS/MS and GC–MS/MS to simulta­ market and field samples to judge the quality of the vegetable foods and
neously determine 73 pesticide residues in okra. The method is short-run other agricultural products. Further, it would support the global food
and reproducible; meeting different regulatory body requirements, standard requirements.
FSSAI, APEDA, EU and Codex Alimentarius Commission tolerance limits
(MRLs less than 10 µgkg− 1 for most of the pesticides). The method had 4.2. Residues in real samples from farm-gate and markets
high accuracy with acceptable recovery at 0.01 mg kg− 1 (as quantifi­
cation limit), less than or equal to EU-MRL. Compared to the previously In the present investigation, 73 agrochemicals were screened in okra
published method on okra and other vegetable matrices, the present fruit for the presence of residue using tandem mass spectrometry. After
investigation has significant findings with respect to recovery, inter and analyzing the field samples, the residues of fenpyroxymate (0.01 mg
intraday precision, matrix effect and simultaneous determination of kg− 1), carbendazim (0.11 mg kg− 1), profenofos (0.02 mg kg− 1), ace­
chemical pesticides. To support the claim, Kumari et al. (2005) reported phate (0.11 mg kg− 1), imidacloprid (0.02 mg kg− 1), hexaconazole (0.03
a method with GC-ECD and NPD to analyze organochlorine, organo­ mg kg− 1), emamectin benzoate (0.01 mg kg− 1), triademenol (0.01 mg
phosphate, synthetic pyrethroids, and carbamates residues in vegetables kg− 1) and bifenthrin (0.02 mg kg− 1) were quantified and confirmed
with limits of detection in the range of 5–100 picograms and this method simultaneously. There is no much difference for the residues observed in
lacks the confirmation of pesticides residues in vegetables. Baig, Akh­ the market sample. Similar compounds were quantified from the market
tera, Ashfaq, and Asi (2009) reported a method for eggplant plants, okra samples; however, the residue level was higher than in field sam­
pumpkins, and okra using HPLC with a UV detector. This method is ples (Table 3 & 4).
having less sensitive, and the technique used is non-confirmatory. Multiresidue analysis in market samples across the globe could
Analysis of multi-residues using HPLC or GC in different vegetable reveal the significant amount of residues of pesticides. A real sample of
foods could be less accurate and acceptability at global food trade for okra from the farm gate market outlets showed substantial residues
export. The tandem mass spectrometry is otherwise an essential tool for when analyzed with the proposed method. However, during 2003, about
quantifying residues in vegetable foods. 20 different market okra samples were analyzed for OC, OP, SP, and
Pesticide residue determination in okra fruits with an analytical carbamates insecticides using a primitive analytical tool viz., GC-ECD
method using GC–MS found 70 to 120% of recovery with 1:1n-hexane and NPD reflected the residues of monocrotophos, malathion, endo­
and dichloromethane extraction (liquid–liquid extraction) with florisil sulfan, and cypermethrin. This study reported the pesticide contami­
clean-up column of okra fruits at 0.10 μg kg− 1 fortification (Essumang, nation of market samples, residues of monocrotophos, and cypermethrin
Asare, & Dodoo, 2013). To quantify pyrethroid residues, the method found above their tolerance level (MRL) (Kumari & John, 2005). Anal­
developed using gas chromatography electron capture detector by ysis of organophosphate pesticide residues in 36 okra samples collected
Chandra (2008) and Kumari et al. (2005) inferior to the method from different farms in Pakistan showed profenophos, triazophos, and
developed on mass spectrometry. Saeid and Selim (2012) standardized a chlorpyriphos in 7, 5, and 1 sample, respectively exceeded the pre­
method to estimate the 86 different crop protection pesticides using scribed MRLs (Baig et al., 2009). Another study involving analysis of 350
GC–MS with a detection limit of 0.01mgkg− 1 for raw non-leafy vegeta­ vegetables (okra, cabbage, tomato, lettuce, carrot, green pepper, onion,
bles. This method reported a high sample size of test portion (120 g) and and cucumber) samples collected from six different markets located at
extraction solvent (200 mL) acetonitrile is absolutely a non-economical Kumasi (Ghana) found to be 19 and 43.5% of the samples had residues
approach. However, it involves a mass technique for the detection and above and below the MRL, respectively (Crentsil, Archibold, Dzifa,

Table 3
Health risk estimation in okra fruits collected at from Field.
Pesticide Residue (mg kg¡1) EDI (mg kg¡1 body weight) ADI (mg kg¡1 body weight) Hazard Index

Children Adult Children Adult

Fenpyroxymate 0.01 0.00442 0.00074 0.01 0.10 0.02

Carbendazim 0.11 0.00071 0.00012 0.03 0.15 0.02
Profenofos 0.02 0.00425 0.00071 0.00 7.13 1.19
Acephate 0.11 0.00071 0.00012 0.03 0.14 0.02
Imidacloprid 0.02 0.00135 0.00022 0.06 0.01 0.00
Hexaconazole 0.03 0.00058 0.00010 0.01 0.27 0.04
Emamectin benzoate 0.01 0.00059 0.00010 0.00 0.29 0.05
Triademenol 0.01 0.00093 0.00015 0.06 0.01 0.00
Bifenthrin 0.02 0.00442 0.00074 0.01 0.09 0.02

EDI-estimated daily intake, ADI-acceptable daily intake,

8
M.S. Pallavi et al. Food Chemistry: X 19 (2023) 100814

Table 4
Health risk estimation in okra fruits collected from the market.
Pesticide Residue (mg kg¡1) EDI (mg kg¡1 body weight) ADI (mg kg¡1 body weight) Hazard Index

Children Adult Children Adult

Metalachlor 0.01 0.00026 0.00004 0.20 0.00 0.00

Carbendazim 0.16 0.00621 0.00103 0.03 0.21 0.03
Profenofos 0.01 0.00040 0.00007 0.00 4.00 0.67
Bifenthrin 0.01 0.00053 0.00009 0.01 0.05 0.01
Acephate 0.04 0.00147 0.00025 0.03 0.05 0.01
Imidacloprid 0.09 0.00349 0.00058 0.06 0.06 0.01
Hexaconazole 0.12 0.00492 0.00082 0.01 0.98 0.16
Emamectin benzoate 0.02 0.00087 0.00014 0.00 0.43 0.07
Fenpyroxymate 0.01 0.00058 0.00010 0.01 0.12 0.02

EDI-estimated daily intake, ADI-acceptable daily intake.

Fig. 3. Ishikawa diagram presenting the uncertainty sources of the measurement uncertainty for pesticide in okra samples analysis.

Fig. 5. LC-MS/MS chromatogram with detected insecticides in sample.

9
M.S. Pallavi et al. Food Chemistry: X 19 (2023) 100814

Fig. 6. Okra market sample chromatogram detected with 1. Bifenthrin, 2. Fenpropathrin, 3. Lambda-Cyhalothrin.

Jacob, & Anita, 2011). Previous multi-residue analysis in vegetable study, the market and farm gate sample analysis detected with multi­
foods reflected the contamination of persistent synthetic pesticides of class pesticides with moderate residues and multiresidue methods are
OC, OP, or SP molecules, and contamination is due to the frequent essential with confirmation techniques such as LC-MS/MS and GC–MS/
application of conventional group insecticides. Organophosphates and MS fulfills requirements of the global food standards.
synthetic pesticides are major contaminants in commonly grown vege­
tables, viz., okra and brinjal. It was observed that the fenitrothion (0.170 5. Conclusions
mg kg− 1) in okra, ethion (10.350 mg kg− 1), acephate (0.363 mg kg− 1),
cypermethrin (0.002 mg kg− 1) and fenitrothion (0.475 mg kg− 1) in In this research, a sensitive and reproducible method was developed
brinjal, respectively (Chowdhury et al., 2014). and validated to simultaneously determine 73 pesticide residues in okra
After 2010, the pesticide contamination in market fruits and vege­ fruit using LC-MS/MS and GC–MS/MS. As per SANTE guidelines, the
tables showed applied chemicals and conventional persistent pesticides. optimized methods provided good precision, precision-intra, and inter­
It was evident from a study of Sheikh et al. (2013) with different day for all the selected pesticides. Further, the laboratory-validated data
vegetable samples found contaminated with organophosphates (chlor­ for measurement uncertainty estimation found a combined uncertainty
pyrifos, profenofos), cyclodine (endosulfan), synthetic pyrethroids (Ux) value in the range of 1.81 to 12.91 µg kg− 1 evaluated at 50 µg kg− 1.
(bifenthrin, and cypermethrin), neonicotinoids (imidacloprid), aver­ The developed method was successfully adopted to screen the organo­
mectin (emamectin benzoate), insect growth regulator (diafenthiuron phosphates, neonicotinoids, fungicides, insect growth regulators resi­
and lufenuron). However, all vegetables had more than one pesticide dues in real okra samples collected from field and market. Out of many
and violated the Japanese MRLs. Among many vegetables, okra is one of pesticides detected in the field and market samples, only a pesticide
the significant vegetables marketed every day in India. It is grown with showed risk to both consumer categories. This method can be employed
multiples pesticides applications, and monitoring the pesticides using for routine monitoring of okra fruits to meet the indigenous and export
the multi-residue method would facilitate the quantum of pesticide requirements. Further, the specific methods can be developed involving
residues on marketable commodities offered for consumption. In similar the pesticides of polar, non polar groups and pesticide which are
studies, the insecticides viz., monocrotophos, chlorpyriphos, and currently used and frequently detected with special reference to the
dimethoate residue analysis in farm gate okra samples showed 42 that, export importance. Methods are also been standardized to shorten the
and 39% of the tested samples contained residues higher residues the analysis time and covering more compounds with lowest level of control
MRL, respectively (Pal et al., 2016). The application of neonicotinoids in in GC–MS/MS and LC-MS/MS.
okra is a common practice in vegetable cultivation. Their high biological
efficacy and frequent application could result in the occurrence of po­ Funding
tential residues. Okra collected from three different vegetable from the
Multan region of Pakistan showed the presence of imidacloprid and This work was conducted as a Faculty Research Programme, Gov­
acetamiprid residues in 58 and 65 % samples, respectively, and with 10 ernment of Karnataka, India, Research Grant Number: COM/UAS/5876/
and 15 % samples found residues above MRLs (Amjad et al., 2019). 2020-21.
Growing vegetables alongside a high pesticide intensive crop yield
higher residues to the adjoining vegetable crops. It was observed that CRediT authorship contribution statement
okra recorded an accumulation of significant residues when planted
close to watermelon. The concentration ranged from 3.10 to 7.60, 2.80 M.S. Pallavi: Methodology, Investigation, Validation, Conceptuali­
to 2016.80, and 0.10 to 4.10 μg kg− 1 for OC, OP, and SP groups, zation, Data curation, Formal analysis, Visualization, Writing – original
respectively, of which the residue levels for methamidophos, malathion, draft, Writing – review & editing. R. Harischandra Naik: Conceptual­
and dimethoate was 6.05, 23.30, and 50.60 μg kg− 1, respectively than ization, Methodology, Investigation, Validation, Visualization, Formal
tolerance limit advocated by WHO/FAO (Essumang et al., 2013). The analysis, Funding acquisition, Project administration, Resources, Su­
peri-urban farming system of Pakistan recorded a high amount of pyre­ pervision, Writing – original draft, Writing – review & editing. K.
throid residues. Synthetic pyrethroid residues have been extensively Pavankumar: Methodology, Investigation, Validation, Formal analysis.
noticed in vegetables and fruits samples in China as well, and it is in the Ratnamma: Investigation, Validation, Formal analysis. Nandini:
range of 1.60 to 1980.00 µgg− 1 (Feng & Jin, 2007). Similarly, a delta­ Investigation, Validation, Formal analysis. A. Shwetha: Investigation,
methrin level was 0.285 mg kg− 1 in okra, 0.306 mg kg− 1 in cauliflower, Validation, Formal analysis. P. Naveenkumar: Investigation, Valida­
and 0.421 mg kg− 1 in spinach (Randhawa et al., 2008). In the present tion, Formal analysis. M. Paramasivam: Conceptualization,

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M.S. Pallavi et al. Food Chemistry: X 19 (2023) 100814

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chromatography mass spectrometry: II-non-leafy vegetables. Journal of Chemistry.
& editing. R. Udaykumar Nidoni: Supervision, Resources, Methodol­
https://doi.org/10.1155/2013/727149
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Supervision, Resources, Funding acquisition, Visualization. M. Bhee­ target crop) grown close to a watermelon farm in Ghana. Environmental Monitoring
manna: Supervision, Resources, Funding acquisition, Project adminis­ and Assessment, 185, 7617–7625. https://doi.org/10.1007/s10661-013-3123-5
European Commission (2008) Regulation (EC) No. 299/2008 of the European Parliament
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on food and feed of plant and animal origin. Off J Eur Commun L 9:67.
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Declaration of Competing Interest
Ferrer, C., Lozano, A., Agüera, A., Jiménez, A., & Fernández, A. R. (2011). Overcoming
matrix effects using the dilution approach in multiresidue methods for fruits and
The authors declare that they have no known competing financial vegetables. Journal of Chromatography A, 1218, 7634–7639. https://doi.org/
10.1016/j. chroma.2011.07.033
interests or personal relationships that could have appeared to influence
FSSAI (2018) Notification-pesticide/stds-FSSAI/2017. Food Safety and Standards
the work reported in this paper. Authority of India. Ministry of Health and Family Welfare, 2018. Available from:
file:///C:/Users/HP/Downloads/ Gazette_Notification_MRL_Pesticides_03_01_2019.
Data availability pdf. Accessed 20.05.2021.
Gupta, S., Sharma, R. K., Gupta, R. K., Sinha, S. R., Singh, R., & Gajbhiye, V. T. (2009).
Persistence of new insecticides and their efficacy against insect pests of okra. Bulletin
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Harischandra, N. R., Pallavi, M. S., Bheemanna, M., PavanKumar, K., Reddy, V. C. S.,
Udaykumar, N. R., … Yadav, S. (2021). Simultaneous determination of 79 pesticides
Acknowledgments in pigeonpea grains using GC–MS/MS and LC–MS/MS. Food Chemistry, 347, Article
128986.
Hingmire, S., Oulkar, D. P., Utture, S. C., Shabeer, T. A., & Banerjee, K. (2015). Residue
The authors gratefully acknowledge the University of Agricultural analysis of fipronil and difenoconazole in okra by liquid chromatography tandem
Sciences, Raichur, India, for research facilities. This research was carried mass spectrometry and their food safety evaluation. Food Chemistry, 176, 145–151.
out under the financial assistance from the Faculty Research Programme Indian Horticulture Database, National Horticulture Board (NHB). 2018-19. J. Hort. Sci.
36(1/2), 175-179.
during the year 2020-21.
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