Académique Documents
Professionnel Documents
Culture Documents
CONTACT S. Karthikeyan karthickacri@yahoo.com Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu 641003, India
© 2018 Taylor & Francis Group, LLC
2 S. KARTHIKEYAN ET AL.
affecting the quality of rice and threatening public (Mumbai, India). Magnesium sulphate and anhy-
health and the environment (Hamsan et al. 2017; drous sodium chloride (heated at 650°C for 4 h
Hladik et al. 2018). In India, rice is the most before use and kept in desiccator) were from
consumed cereal; eaten cooked and also as pro- Merck India Ltd. (Mumbai, India) Primary sec-
cessed products such as puffed rice, popped rice ondary amine (PSA) and graphitized carbon black
and flaked rice. Though more focus is given on (GCB) were purchased from M/s. Agilent
residues in grain, their presence in straw goes Technologies (Agilent Technologies India Pvt
neglected. Paddy straw is being used as major Ltd, Chennai, India). Merck Millipore system
livestock fodder in the whole of South India, was used to collect ultra-pure water. Nylon filters
although it is mainly composed of cellulose, hemi- (0.2 µm) from PAL life sciences (PALL life
celluloses and lignin (Yang et al. 2007; Matano sciences, Bengaluru, India) were used to filter the
et al. 2014). Livestock are the main source of sample extracts.
proteins and minerals as meat and milk for the
Indian subcontinent (Randolph et al. 2007;
Preparation of standard solutions
Lammers et al. 2009). Straw may be one of the
routes of exposure of animals to pesticides. Stock solutions (1000 µg mL−1) of the five neoni-
Already, neonicotinoid residues have been cotinoid compounds were prepared individually
reported in the nestlings of Eurasian eagle owl by dissolving the technical-grade material in acet-
(Taliansky-Chamudis et al. 2017) and also in onitrile (v/v) separately. These were labelled and
honey (Mitchell et al. 2017). stored in a freezer at –20°C. From the stock solu-
Thus, monitoring pesticide residues in rice tion, intermediate stock and working standard
whole grain and rice straw is imperative as far as solutions were prepared as mixes.
public human health is concerned. Several studies
have reported the methodologies for determina-
Sample preparation
tion of neonicotinoids in honey, food grain, fruit
and vegetables (Takatori et al. 2008; Mastovska The rice whole grain and rice straw samples were
et al. 2009; Kamel 2010; Liu et al. 2010). collected from organic demo fields of Department
However, few reports are available on methodol- of Sustainable Organic Agriculture, Tamil Nadu
ogies for the determination of neonicotinoids in Agricultural University (TNAU), Coimbatore,
rice grain, rice hull and rice straw (Zhang et al. Tamil Nadu, India. Rice grains along with hull
2017). In this study, attempts were made to sim- (whole grains) were homogenized into fine pow-
plify the extraction and clean up procedures from der using blender. Rice straws were milled into
an existing method. Particularly, to our knowl- coarse powders using a Wiley mill at Department
edge, no studies have been reported for multi- of Forage and Fodder crops, TNAU and were used
residue analysis of neonicotinoids in rice whole after passing through 0.1 mm metal sieves.
grain and rice straw using LC-MS/MS. Hence,
this study was undertaken to develop a method Extraction and clean up
for the analysis of neonicotinoid insecticide resi- The QuEChERS method (Anastassiades et al.
dues using a modified QuEChERS method in rice 2003) with following modifications was employed
whole grain and rice straw and to determine the for extraction and clean-up process. Homogenized
residues in farm-collected samples. rice whole grain powder (10 g) was weighed out
into a 50-mL centrifuge tube. After adding 20 ml
of acetonitrile, the centrifuge tube was capped and
Materials and methods
vortex shaken for 1 min. To this, 4 g anhydrous
Certified pesticide standards (>90%) of acetami- magnesium sulphate and 1 g of sodium chloride
prid, thiamethoxam, thiacloprid, imidacloprid, was added and again vortex shaken for 1 min to
and clothianidin, and solvents and reagents mix the sample thoroughly. The samples were
including acetonitrile and formic acid of MS centrifuged for 10 min at 6000 rpm. From this,
grade were obtained from Sigma Aldrich 9 mL of supernatant was passed through 4 g of
FOOD ADDITIVES & CONTAMINANTS: PART A 3
anhydrous sodium sulphate to remove moisture (4.8 × 250 mm) column with a mobile phase of
and out of this, 6 mL of extract was transferred acetonitrile: water acidified with 0.5% formic acid
into centrifuge tube containing 100 mg PSA and (50:50, v/v) at 0.5 mL min−1 flow rate. With these
600 mg anhydrous magnesium sulphate. The sam- conditions, all the five analytes were detected in
ples were mixed thoroughly by vortexing for 10.0 min. Confirmation was done utilizing
1 min and centrifuged for 10 min at 3000 rpm. Acquity (Waters, USA) Tandem Quadrupole
Finally, 4 mL of acetonitrile layer was transferred mass Detector (TQD) with Electrospray ionization
into a clean glass test tube and concentrated to (ESI) interface. All the five analytes were ionized
near dryness using nitrogen gas in a Turbovap in positive polarity. The standardized ESI condi-
(Caliper life sciences, USA) at 40°C. The final tions were as follows: capillary voltage at 3.5 kV;
volume was reconstituted to 1 ml using acetoni- source block and desolvation temperatures were
trile and transferred into a 1.5 mL glass auto 150°C and 500°C, respectively, desolvation and
sampler vial for LC/MS-MS analysis after filtering cone gas flows were fixed at 1100 and 50 L h−1,
through a 0.2 µm filter membrane. respectively, and the collision gas flow was
For straw sample analysis, rice straw powder (2 g) 0.18 mL min−1. Nitrogen was used for desolvation
was taken into a 50 mL centrifuge tube. To the purpose and also as cone gas, while the collision
sample, 5 mL distilled water was added and shaken gas was argon. Infusion was done at 20 μL min−1
well to moisten the sample. This was followed by and the lens voltages were standardized using
addition of 20 mL of acetonitrile. The samples were Tune Master (Mass Lynx software). For infusion
homogenized using silent crusher (Make: Heidolph, while tuning, working standards of 0.5 and
Germany) for 5 min at 15,000 rpm. After adding 4 g 1.0 μg mL−1 were used. Multiple reaction moni-
anhydrous magnesium sulphate and 1 g of sodium toring mode was followed and the neonicotinoids
chloride, the centrifuge tube was vortex-shaken for were detected as reported in Table 1.
1 min and centrifuged for 10 min at 6000 rpm. From
the supernatant, 9 ml was taken and dehydrated
using 4 g of anhydrous sodium sulphate, and again Method validation
6 ml of the extract was transferred into an 18-mL The efficacy of the developed method was proved
centrifuge tube containing 10 mg GCB, 100 mg PSA by assessment of specificity, linearity, limits of
and 600 mg anhydrous magnesium sulphate. The detection and quantification, precision and accu-
extract was mixed vigorously by vortexing for 1 min racy. Organic rice samples with no residues were
and centrifuge-extracted for 10 min at 3000 rpm. An used for all the analyses.
aliquot of 4 mL of the supernatant was transferred to The linearity of neonicotinoids was studied
Turbovap tube, dried under nitrogen gas and recon- using five linear concentrations of the mix solu-
stituted to 1 mL with acetonitrile. This extract was tion in the range of 0.025–0.25 μg mL−1. The
transferred into 1.5 mL glass auto sampler vial for calibration standards were prepared using 100%
LC/MS-MS analysis after filtering through 0.2 µm acetonitrile. Three replicates were made for each
filter membrane. concentration against which the detector’s
responses were plotted. Regression equation with
slope, y-intercept and coefficient of correlation
Instrumentation
were evaluated for the five neonicotinoids. Matrix-
The chromatographic separation was carried out matched standards were also studied to prove that
in a Waters Alliance system using C18, 5 µm there is no matrix effect. The LOD were calculated
Table 2. Linearity of rice whole grain and rice straw matrix match neonicotinoid standards.
Rice grain Rice straw
Analyte Concentration range Calibration curve R2 value Calibration curve R2 value
Acetamiprid 0.025–0.25 189.156x + (−920.366) 0.981576 230.444x + 812.18 0.993600
Thiacloprid 0.025–0.25 415.58x + (−1646.3) 0.989100 397.135x + 1580.7 0.995023
Imidacloprid 0.025–0.25 34.8896x + (−146.474) 0.983934 39.2176x + 44.5258 0.991208
Thiamethoxam 0.025–0.25 34.5698x + (−115.568) 0.993438 30.393x + 20.035 0.984057
Clothianidin 0.025–0.25 10.9306x + (−40.2987) 0.978965 12.2628x + 129.183 0.982724
Discussion
Figure 2. LC-MS/MS chromatogram of thiamethoxam for rice In the present study, a modified QuEChERS sam-
whole grain matrix-matched standard (0.025 mg kg−1). ple preparation procedure for LC-MS/MS analysis
was validated to determine neonicotinoid residue
in rice whole grain and rice straw. Studies shown
reported that in plant samples of cereals such as
rice, wheat and oats, the organic contents that are
co-extracted along with the residues act as inter-
ferences in the chromatograms (Schwedler et al.
2000; Koesukwiwat et al. 2010). However, in this
study, interferences were overcome by improved
clean up step. The samples were extracted using
acetonitrile and the dispersive solid-phase clean-
up procedure was followed effectively to remove
the interfering co-extractives from grain and straw
samples. In case of rice straw, while coloured
extract was obtained due to the chlorophyll con-
Figure 3. LC-MS/MS chromatogram of thiamethoxam for rice tent, this was removed by using GCB in the clean-
straw matrix-matched standard (0.025 mg kg−1).
up step. Recovery above 70% and RSD below 20%
were obtained for rice whole grains. In case of rice
grains and rice straw, respectively (Tables 3 and straw, the recovery at/above the level of 60% at
4). The intraday precision observed in terms of few concentrations may be due to interference by
RSD ranged between 3.35% and 17.55%, and organic matters but acceptable as the RSD was less
2.19% and 10.31% for grain and straw, than 20%. The results indicate that the present
respectively. method has a good recovery rate within the cri-
Monitoring of farm gate samples comprising teria set by SANTE (2017) for both rice whole
conventional field, IPM and organic fields samples grain and rice straw.
of two crop seasons by the validated method MRLs for neonicotinoid pesticides are not set
revealed that one sample of rice whole grain, out for rice whole grains (with hull) and rice straw
of 24 samples, was contaminated with under Food Safety and Standards Authority of
6 S. KARTHIKEYAN ET AL.
Table 3. Recovery, LOD and LOQ of neonicotinoid insecticide residues in paddy grains.
Spiked level (mg kg−1)
0.025 0.063 0.125 0.187 0.25
LOD LOQ
Pesticides Recovery (%) RSD Recovery (%) RSD Recovery (%) RSD Recovery (%) RSD Recovery (%) RSD (mg kg−1) (mg kg−1)
Acetamiprid 76.10 12.30 91.76 6.81 96.84 15.01 103.99 8.18 110.18 5.65 0.007 0.024
Thiacloprid 88.67 12.13 90.78 6.47 107.94 17.55 115.70 8.87 100.01 6.66 0.0084 0.028
Imidacloprid 74.92 12.67 90.68 8.00 96.59 16.11 104.19 11.37 91.05 6.26 0.008 0.025
Thiamethoxam 80.68 12.92 91.43 7.70 96.56 11.84 111.18 10.21 87.29 4.56 0.008 0.027
Clothianidin 87.75 10.76 89.49 6.22 92.63 10.41 100.96 8.44 79.01 3.35 0.007 0.024
Table 4. Recovery, LOD and LOQ of neonicotinoid insecticide residues in paddy straw.
Spiked level (mg kg−1)
0.025 0.063 0.125 0.187 0.25
LOD LOQ
Pesticides Recovery (%) RSD Recovery (%) RSD Recovery (%) RSD Recovery (%) RSD Recovery (%) RSD (mg kg−1) (mg kg−1)
Acetamiprid 82.09 9.60 68.86 2.19 68.77 4.22 75.51 9.50 74.71 2.74 0.006 0.021
Thiacloprid 80.50 7.68 68.46 3.28 69.84 3.83 77.71 7.61 75.50 3.71 0.005 0.016
Imidacloprid 104.81 7.94 73.94 3.95 71.95 10.31 85.15 9.86 74.56 3.70 0.015 0.050
Thiamethoxam 84.09 8.14 70.29 6.23 69.42 10.05 78.81 10.25 74.42 6.18 0.01 0.038
Clothianidin 80.58 4.58 60.00 7.65 60.01 6.81 69.41 4.56 62.55 3.37 0.009 0.031
black-streaked dwarf virus in insect vector. J Virol Takatori S, Okihashi M, Okamoto Y, Kitagawa Y,
Methods. 211:32–35. Kakimoto S, Murata H, Sumimoto T, Tanaka Y. 2008.
Liu S, Zheng Z, Wei F, Ren Y, Gui W, Wu H, Zhu G. 2010. A rapid and easy multiresidue method for the determina-
Simultaneous determination of seven neonicotinoid pesti- tion of pesticide residues in vegetables, fruits, and cereals
cide residues in food by ultraperformance liquid chroma- using liquid chromatography/tandem mass spectrometry.
tography tandem mass spectrometry. J Agric Food Chem. J AOAC Int. 91(4):871–883.
58(6):3271–3278. Taliansky-Chamudis A, Gómez-Ramírez P,
Mastovska K, Dorweiler KJ, Lehotay SJ, Wegscheid JS, León-Ortega M, García-Fernández A. 2017. Validation
Szpylka KA. 2009. Pesticide multiresidue analysis in cereal of a QuECheRS method for analysis of neonicotinoids
grains using modified QuEChERS method combined with in small volumes of blood and assessment of exposure
automated direct sample introduction GC-TOFMS and in Eurasian eagle owl (Bubo bubo) nestlings. Sci Total
UPLC-MS/MS techniques. J Agric Food Chem. 58 Environ. 595:93–100.
(10):5959–5972. Teló GM, Senseman SA, Marchesan E, Camargo ER, Jones T,
Matano C, Meiswinkel TM, Wendisch VFI. 2014. Amino McCauley G. 2015. Residues of thiamethoxam and chlor-
acid production from rice straw hydrolyzates. In: antraniliprole in rice grain. J Agric Food Chem. 63
Watson RR, Preedy VR, Zinadi S, editors. Wheat and (8):2119–2126.
rice in disease prevention and health. San Diego (CA): Tomizawa M, Casida JE. 2003. Selective toxicity of neoni-
Academic Press; p. 493–505. cotinoids attributable to specificity of insect and mam-
Matsuda K, Buckingham SD, Freeman JC, Squire MD, malian nicotinic receptors. Annu Rev Entomol. 48
Baylis HA, Sattelle DB. 1998. Effects of the α subunit on (1):339–364.
imidacloprid sensitivity of recombinant nicotinic acetyl- Tomizawa M, Lee DL, Casida JE. 2000. Neonicotinoid insec-
choline receptors. Br J Pharmacol. 123(3):518–524. ticides: molecular features conferring selectivity for insect
Mishra AK, Kumar A, Joshi PK, D’Souza A, Tripathi G. versus mammalian nicotinic receptors. J Agric Food
2018. How can organic rice be a boon to smallholders? Chem. 48(12):6016–6024.
Evidence from contract farming in India. Food Policy. Van Hoed V, Depaemelaere G, Ayala JV, Santiwattana P,
75:147–157. Verhé R, De Greyt W. 2006. Influence of chemical refining
Mitchell EA, Mulhauser B, Mulot M, Mutabazi A, Glauser G, on the major and minor components of rice brain oil.
Aebi A. 2017. A worldwide survey of neonicotinoids in J Am Oil Chem Soc. 83(4):315–321.
honey. Science. 358(6359):109–111. Yadav M, Prasad R, Kumar P, Pandey C, Prasad D, Kumar P.
Randolph T, Schelling E, Grace D, Nicholson CF, Leroy J, 2018. Effect of date of transplanting on the incidence of
Cole D, Demment M, Omore A, Zinsstag J, Ruel M. 2007. green leaf hopper (GLH), Nephotettix virescens (Distant)
Invited review: role of livestock in human nutrition and & N. nigropictus (Stal) in rice field, Jharkhand.
health for poverty reduction in developing countries. J Pharmacogn Phytochem. 7(SP1):897–900.
J Anim Sci. 85(11):2788–2800. Yamamoto I, Tomizawa M, Saito T, Miyamoto T,
SANTE. 2017. https://ec.europa.eu/food/sites/food/files/plant/ Walcott EC, Sumikawa K. 1998. Structural factors contri-
docs/pesticides_mrl_guidelines_wrkdoc_2017-11813.pdf buting to insecticidal and selective actions of
Schwedler DA, Thomas AD, Yeh L-T. 2000. Determination of neonicotinoids. Arch Insect Biochem Physiol Published
spinosad and its metabolites in food and environmental Collab Entomol Soc Am. 37(1):24–32.
matrices. 2. Liquid chomatography−mass spectrometry. Yang H-Y, Gao L-J, Wang X-F, Wang W-D, Cui Z-J. 2007.
J Agric Food Chem. 48(11):5138–5145. Effects of cultivation conditions on the diversity of
Simon-Delso N, Amaral-Rogers V, Belzunces LP, microbes involved in the conversion of rice straw to
Bonmatin J-M, Chagnon M, Downs C, Furlan L, fodder. J Environ Sci. 19(1):67–73.
Gibbons DW, Giorio C, Girolami V. 2015. Systemic insec- Zhang F, Li Y, Yu C, Pan C. 2012. Determination of six
ticides (neonicotinoids and fipronil): trends, uses, mode of neonicotinoid insecticides residues in spinach, cucum-
action and metabolites. Environ Sci Pollut Res. 22(1):5–34. ber, apple and pomelo by QuEChERS method
Srikaeo K. 2014. Organic rice bran oils in health. In: Watson RR, and LC–MS/MS. Bull Environ Contam Toxicol. 88
Preedy VR and Zibadi S, editors. Wheat and rice in disease (6):885–890.
prevention and health. London: Elsevier; p. 453–465. Zhang Z, Zheng Z, Zhu G, Yu X, Wang D, Liu X. 2017.
Suganthi A, Bhuvaneswari K, Ramya M. 2018. Determination Validation of analytical method and evaluation of clothia-
of neonicotinoid insecticide residues in sugarcane juice nidin residues in rice in a typical Chinese field ecosystem.
using LCMSMS. Food Chem. 241:275–280. J Agric Sci. 155(9):1371–1380.