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Validation and Uncertainty Analysis of a Multiresidue Method for
67 Pesticides in Made Tea, Tea Infusion, and Spent Leaves Using Ethyl
Acetate Extraction and Gas Chromatography/Mass Spectrometry
BAPPADITYA KANRAR, SUDEB MANDAL, and ANJAN BHATTACHARYYA
1
Bidhan Chandra Krishi Viswavidyalaya, Department of Agricultural Chemicals, Export Testing Laboratory,
Mohanpur-741252, West Bengal, India
A rapid, specific, and sensitive multiresidue
method to determine 67 pesticides in made tea, tea
infusion, and spent leaves was developed and
validated for routine analysis by GC/MS with an
approximately 29 min GC run time. The method
was reproducible (HorRat <0.5 at 50 ng/g) when
validated at 50 and 100 ng/g. The samples were
extracted with ethyl acetate–cyclohexane (9 + 1,
v/v), and the extracts were cleaned up by
dispersive SPE with primary-secondary amine
sorbent + graphitized carbon black + Florisil. The
recoveries of all the pesticides were within
70–120% with an RSD of <20% at 50 ng/g and
R2
> 0.99. The matrix effect on the signals of the
compounds was corrected by using
matrix-matched calibration standards. The LOQ
met the requirements of the maximum residue
limits for pesticides in tea as recommended by the
European Union.
T
ea is a popular beverage throughout the world and is
valued for its specific aroma and flavor as well as its
health-promoting properties (1). India is one of the
major tea-producing countries in the world. Among the
factors limiting the quality and quantity of tea production, the
role of insect pests is important. Management of pests in tea
plantations largely depends on the use of broad-spectrum
synthetic chemical pesticides, viz., organophosphates,
carbamates, synthetic pyrethroids, and neonicotinoids, etc. In
recent years, a number of research papers have dealt with the
behavior of different pesticides in tea, focusing on the
influence of various manufacturing processes on the residues
in made tea and their transfer potential to infusion (2, 3).
Residue levels of many pesticides in made tea and its infusion
have also been reported (4–8).
Trace-level multiresidue analysis of pesticides in tea has
become important because of the increasingly stringent
regulatory requirements of the European Union (EU) agencies
and other tea-importing countries (9). In general, pesticide
residue analysis is carried out in a sequence of steps, viz.,
extraction of target compounds from the sample matrix,
cleanup and preconcentration, and, finally, chromatographic
analysis (10, 11). Pesticide residue analysis methods have been
widely developed to analyze multiresidues in fresh vegetables,
fruit, water, honey, etc. (12). Cai et al. (13) applied
polyphenylmethylsiloxane as a coating for solid-phase
microextraction combined with microwave-assisted extraction
to determine the concentrations of organochlorine pesticides in
Chinese tea. The extracts were analyzed by GC with an electron
capture detector. Huang et al. (14) used acetone–ethyl
acetate–hexane for the extraction of pesticides, gel permeation
chromatography (GPC) and SPE for cleanup, and GC/MS
under retention time-locked conditions for the determination of
102 pesticide residues in tea. Yang et al. (15) proposed the
extraction of tea with ethyl acetate–hexane (1 + 3, v/v), cleanup
by GPC and SPE, and subsequent identification and
quantification of selected pesticides by GC/MS.
A literature survey revealed the lack of a suitable
cost-effective multiresidue method (MRM) for trace-level
quantification of pesticide residues in tea matrix. Mastovska
and Lehotay (16) compared the suitability of six organic
solvents for pesticide residue analysis and the stability of
multiclass pesticides, and they identified acetonitrile as the
most suitable extraction solvent for a variety of matrixes.
Ethyl acetate is equally acceptable as an extraction solvent for
different products (17–19), since it does not pose limitations
in terms of lipid coextractives.
The aim of this paper was to optimize and validate a
multiresidue analysis method based on ethyl
acetate–cyclohexane extraction followed by simultaneous
determination of 67 pesticides in tea by GC/MS with good
selectivity, high sensitivity, and a wide application scope.
Experimental
Apparatus
(a) GC/MS instrument.—The extracts were analyzed with
a Varian (Walnut Creek, CA) Saturn 2200 mass spectrometer
coupled to a Model 3800 gas chromatograph. The mass
spectrometer was used in the full-scan mode with electron
KANRAR ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 2, 2010 411
Guest edited as a special report on “Novel GC/MS, HPLC/MS, and
HPLC-DAD-Based Methods for Determination of Pesticide Residues in
Food, Feed, Water, and Soil Samples” by Kaushik Banerjee.
1
Corresponding author’s e-mail: anjan_84@rediffmail.com

412 KANRAR ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 2, 2010
Table 1. Parameters for the analysis of the selected pesticides by GC/MS
Pesticides RT, mina MW
m/z for
quantification Q1
b Q2
b
LOQ,
ng/gc
LOQ,
ng/gd
LOQ,
ng/ge
MRL,
mg/kgf
Carbofuran 3-keto 7.53 236 178 177 — 5 8 10 0.05
Phorate 9.88 260 75 121 97 15 25 30 0.1
a-HCH 10.08 288 183 181 219 5 7 10 0.02g
Fenpyroxymate 10.39 421 213 77 142 6 8 10 0.1
b-HCH 10.65 288 183 181 219 4 5 6 0.02g
g-HCH 10.79 288 183 181 219 5 6 9 0.05
Fluchloralin 10.87 355 306 326 63 2 3 3 —
Chlorothalonil 11.16 266 266 264 267 2 2 3 0.1
d-HCH 11.47 288 183 181 219 4 5 5 0.02g
Cartap hydrochloride
h
11.62 274 149 103 70 2 2 3 0.1
Dichlorvos 11.63 220 109 185 79 3 3 3 0.1
Acetochlor 11.91 270 223 146 59 5 5 6 0.01
Alachlor 12.21 270 188 160 146 3 7 11 0.01
Oxadiargyl 13.06 341 149 — — 17 20 21 0.05
Malathion 13.27 330 173 125 93 9 11 17 0.05
Chlorpyrifos 13.53 349 314 197 97 1 2 2 0.1
Phorate sulfone 13.53 292 97 + 125 153 65 6 13 21 0.1i
Phorate sulfoxide 13.29 276 199 + 125 97 153 7 19 28 0.1i
Aldrin 13.57 362 66 263 79 16 16 18 0.02j
Thiobencarb 13.62 258 100 125 72 4 3 4 —
Tetraconazole 13.87 372 336 — 5 4 5 0.02
Dicofol 14.22 370 139 251 141 4 4 4 20
Pendimethalin 14.80 281 252 162 191 4 4 5 0.1
Fipronil 15.00 437 367 369 — 2 3 5 —
o,p¢-DDE 16.169 316 318 246 248 2 2 3 —
Paclobutazole 16.38 294 238 236 125 10 11 12 —
Butachlor 16.48 311 176 + 160 57 — 6 8 13 —
a-Endosulfan 16.63 404 241 + 239 265 — 6 9 10 30k
Hexaconazole 17.16 314 214 216 82 10 9 10 0.05
Pretilachlor 17.21 311 238 162 176 2 2 2 —
Metomino strobin 17.39 284 191 + 196 238 — 2 2 3 —
p,p¢-DDE 17.65 316 318 246 248 2 2 2 0.2l
Dieldrin 17.72 381 79 81 77 12 18 25 0.02j
Tricyclazole 17.76 189 189 162 135 25 27 41 —
o,p¢-DDD 17.79 318 235 237 165 3 2 4 0.2l
Oxyfluorfen 17.84 362 252 361 300 4 4 5 0.05
Buprofezin 17.84 305 105 + 106 + 172 83 104 10 15 20 0.05
Flusilazole 17.89 315 233 + 234 206 315 12 14 10 0.05
Chlorfenapyr 18.25 408 364 328 — 5 4 5 50
Endosulfan alcohol 18.85 361 69 + 229 259 237 40 45 50 —
Chromafenozide 18.906 395 175 — — 40 50 50 —
b-Endosulfan 19.02 404 241 + 195 + 159 239 243 25 40 40 30k
Ethion 19.21 384 231 153 125 4 3 4 3
p,p¢-DDD 19.23 318 235 237 165 2 2 2 0.2l
o,p¢-DDT 19.27 352 235 237 165 2 2 2 0.2l

impact ionization. The system was equipped with a
Model 1079 programmed temperature vaporizer injector,
electronic flow control, and autosampler (CTC Combipal).
The injection liner (single-gooseneck, 3.4 mm id) contained a
plug of Carbofrit (Restek, Bellefonte, PA) to allow 8 mL
injections of the ethyl acetate extracts. Varian MS
Workstation software (Version 5.1.2600.2180) was used for
instrument control and data analysis.
(b) Low-volume concentrator.—The samples were
evaporated using a Turbo Vap LV from Caliper Life Science
(Hopkinton, MA).
(c) Centrifuge.—The extracts were centrifuged using a
high-speed, refrigerated centrifuge, Model Avanti J-30I
(Beckman Coulter, Fullerton, CA). The rotor heads were
suitable for holding eight 50 mL (JA-30.50 T1) and eighteen
10 mL (JA-21) tubes.
(d) Analytical balance.—A top-loading balance with
digital display (Sartorius CP 225D; Goettingen, Germany)
was used to weigh the samples and powder reagents.
(e) Vials and vessels.—For both the extraction and
dispersive SPE (d-SPE) cleanup steps, fluorinated ethylene
propylene (FEP) centrifuge tubes (Nalgene, Rochester, NY)
Table 1. (continued)
Pesticides RT, mina MW
m/z for
quantification Q1b Q2b
LOQ,
ng/gc
LOQ,
ng/gd
LOQ,
ng/ge
MRL,
mg/kgf
Triazophos 19.83 313 161 77 97 25 28 35 —
Benalaxyl M 20.17 325 148 206 — 4 5 5 0.1
Endosulfan sulfate 20.33 420 387 272 237 16 16 20 30k
Propiconazole 20.58 342 259 69 173 20 20 22 —
p,p¢-DDT 20.688 352 235 237 165 6 5 6 0.2l
Tebuconazole 21.07 307 250 125 70 15 22 26 0.05
Propargite 21.12 350 173 135 81 9 10 13 5
TPP (IS) 21.25 326 325 326 — — — — —
Carbosulfan 21.87 380 160 118 135 20 21 25 0.1
Bifenthrin 22.18 422 181 166 165 4 4 4 5
Anilophos 22.67 368 226 334 228 16 16 20 —
Fenazaquin 22.70 306 145 144 160 7 6 7 0.01
Tetradifen 23.12 356 356 + 159 + 111 229 227 50 48 50 0.05
Phosalone 23.15 378 182 184 — 13 16 17 0.1
Cyhalofop butyl 23.50 357 357 256 229 25 25 30 0.05
Amitraz 23.63 293 162 121 147 17 20 25 0.1
l-Cyhalothrin 23.67 449 197 181 208 6 5 6 1
a-Cypermethrin 26.09 415 181 183 185 10 22 25 0.5
Etofenprox 26.20 376 163 164 — 9 10 13 0.01
Fenvalerate I+II 26.99, 27.28 419 125 + 225 167 — 25 35 40 0.05
G-Fluvalinate 27.13 503 250 + 181 252 55 30 37 42 —
Difenoconazole 27.82 406 323 325 152 40 50 50 0.05
Deltramethrin 27.86 503 253 181 77 20 45 50 5
a
RT = Retention time.
b
Q1 and Q2 are the qualifier ions.
c
LOQ in tea infusion.
d
LOQ in spent leaves.
e
LOQ in made tea.
f
MRL established by EU Regulation (EC) No. 396/2005 updated on 05/20/2009.
g
Sum of HCH isomers except g isomer.
h
Quantified as nereistoxin.
I
Sum of phorate, is oxygen analogs and their sulfones expressed as phorate.
j
Sum of aldrin and dieldrin.
k
Sum of a, b isomers and endosulfan sulfate.
l
Sum of p,p¢-DDT; o,p¢-DDT; p,p¢-DDE; and p,p¢-DDD.

noisufniaetdna,aetedam,sevaeltnepsmorfsedicitseptsetehtfoEMdna,taRroH,DSR,yrevoceR.2elbaT
edicitseP
faeltnepSaetedaMnoisufniaeT
g/gn05ag/gn001ataRroHb%,EMcg/gn05ag/gn001ataRroHb%,EMcg/gn05ag/gn001ataRroHb%,EMc
otek-3narufobraC)50.5(43.89)64.01(71.5902.083.5–)00.01(0.001)79.11(71.6904.083.4–)33.8(66.201)46.6(76.10133.067.3–
etarohP)24.8(43.77)47.5(76.5743.064.0)22.9(0.97)94.5(38.6773.067.9)56.6(66.18)04.9(71.0872.031.8
aHCH-)04.01(66.28)55.5(71.8724.011.42–)51.21(66.48)14.4(71.9794.041.32–)20.21(43.78)78.9(05.2884.072.6–
etamyxorypneF)73.21(43.68)06.21(00.9905.035.31–)62.11(0.98)04.51(76.00154.045.81–)40.01(0.29)05.11(38.30104.015.8–
b-HCH)94.11(43.48)34.41(23.1964.088.4–)20.21(66.68)37.41(52.3984.068.2–)09.11(43.98)01.51(78.4984.083.6–
gHCH-)44.21(0.19)30.9(26.6905.093.6–)30.21(0.39)87.01(31.8984.029.4–)25.9(0.69)61.01(27.9983.087.21–
nilarolhculF)20.11(0.38)12.9(71.6944.014.8–)90.9(0.68)73.9(05.7973.041.7–)10.01(66.98)66.01(00.9904.026.7–
linolahtorolhC)12.01(0.58)98.01(38.0714.075.91)24.11(66.88)13.01(71.2764.064.32)64.11(0.19)51.01(76.3764.061.72
dHCH-)13.9(66.801)95.7(05.11173.022.9–)13.21(66.111)86.9(76.31194.024.7–)97.11(0.411)03.8(32.51174.024.9–
patraC
edirolhcordyh
)53.01(66.97)34.31(71.2924.068.31–)16.11(0.28)59.31(33.3974.077.21–)41.21(66.48)78.51(00.6894.012.11–
sovrolhciD)20.11(66.38)42.11(71.8744.084.53–)48.01(43.58)50.21(71.9744.056.43–)97.9(66.88)30.9(33.1893.066.03–
rolhcotecA)89.01(43.07)24.8(05.7744.066.71–)09.8(66.07)67.9(00.8763.031.71–)28.4(0.37)71.6(00.0891.088.21–
rolhcalA)15.11(66.27)05.11(33.7864.046.7–)12.01(43.17)78.01(05.9814.053.5–)12.11(60.47)06.8(00.2954.043.41–
lygraidaxO)94.8(66.57)56.3(76.3743.072.42)50.21(43.87)52.4(05.5784.063.72)98.01(66.18)66.4(38.7744.020.31
noihtalaM)91.11(43.201)89.11(51.80154.029.9–)15.11(66.401)60.21(34.90164.078.6–)76.9(66.701)65.01(89.01193.076.12–
sofiryprolhC)10.21(66.77)59.11(76.4984.089.1–)30.9(0.38)64.21(51.6963.044.0–)80.8(43.68)38.01(09.7923.099.11–
enoflusetarohP)59.11(43.08)59.11(00.0884.080.41–)37.8(40.28)16.21(76.0853.063.31–)96.11(7.38)54.11(71.2874.034.64–
edixoflusetarohP)28.6(43.47)45.31(76.4772.015.72–)65.7(43.67)37.31(75.5703.036.62–)98.4(02.08)35.01(70.7702.063.55–
nirdlA)58.11(0.87)30.81(33.5784.080.8–)90.11(0.08)08.94(38.6754.052.6–)25.21(66.08)76.51(04.8705.038.32–
bracneboihT)97.11(0.67)97.9(89.6874.017.6–)60.21(64.97)97.01(71.7884.015.6–)49.11(8.28)72.11(53.8884.081.4–
elozanocarteT)80.9(0.28)65.01(01.9963.001.21–)64.01(43.48)13.31(50.20124.094.9–)16.7(0.88)91.21(71.7813.069.06–
lofociD)16.11(66.38)93.9(23.3874.095.11–)30.11(66.58)20.01(27.4844.001.01–)24.01(43.88)22.8(24.6824.047.8–
nilahtemidneP)01.21(43.49)47.01(72.30194.046.5–)50.01(43.79)82.01(78.30104.099.1–)45.21(43.89)71.9(39.40105.009.0–
linorpiF)21.21(0.97)04.6(05.9794.013.22–)12.11(66.08)21.7(71.1854.026.03–)85.11(66.18)20.9(38.3874.044.83–
p,o¢EDD-)27.6(66.58)62.9(74.4872.088.01–)48.7(66.78)20.11(28.5813.064.9–)02.7(0.09)79.9(59.9892.001.6–
elozatubolcaP)36.9(43.07)95.41(76.6893.072.62–)10.21(43.27)56.51(38.7884.082.52–)63.01(66.57)10.51(33.1924.070.41–
rolhcatuB)17.8(43.09)19.51(08.8853.068.62)87.8(66.29)98.11(53.0953.070.92)05.9(0.69)96.41(80.3983.061.91
anaflusodnE-)36.01(66.38)60.4(75.9834.019.0–)86.9(0.68)21.6(38.0993.094.5–)43.4(66.09)08.4(89.2971.057.3–
elozanocaxeH)87.7(43.69)51.01(05.8913.080.5–)73.21(0.29)22.01(89.8905.026.4–)48.11(67.89)13.12(57.00184.046.3–

(.2elbaTdeunitnoc)
edicitseP
rolhcaliterP)48.01(0.07)46.41(55.5744.038.42–)15.11(43.07)24.41(30.7764.053.32–)13.21(66.17)23.41(87.8794.020.23–
nibortsonimoteM)44.9(43.07)28.01(88.0783.074.92–)21.01(66.07)49.11(85.2714.087.72–)07.8(43.07)83.9(26.5753.091.14–
elozalcycirT)52.21(0.19)68.51(23.9994.051.32–)20.11(66.39)11.81(83.9944.077.9–)56.9(0.001)33.31(53.30193.088.82–
p,p¢EDD-)52.9(66.38)88.5(05.5873.099.5–)17.6(06.68)27.5(58.6872.015.4–)09.8(49.98)29.7(56.8863.057.62–
nirdleiD)57.01(43.87)86.9(77.9834.076.42–)13.01(0.07)29.5(02.2714.000.9–)22.8(0.57)18.7(32.4733.060.1–
p,o¢-DDD)71.6(43.09)12.01(07.6852.099.02–)50.6(43.29)65.11(81.7842.018.23–)43.7(0.69)60.21(01.9892.040.82–
nefroulfyxO)11.21(0.001)19.31(36.7994.070.51–)47.11(66.101)50.41(86.7974.032.51–)73.6(43.89)98.31(34.9962.003.6–
nizeforpuB)40.21(0.27)10.41(76.1894.078.81–)15.01(66.37)42.21(33.3824.022.71–)12.21(4.67)46.9(05.6894.053.62–
elozalisulF)31.21(5.401)68.01(00.80194.055.7–)43.21(3.701)95.21(71.11105.052.8–)09.11(9.901)55.21(38.31184.057.02–
rypanefrolhC)27.8(0.98)96.41(00.60153.004.9–)29.5(0.19)34.41(36.70142.010.8–)26.9(2.39)20.51(79.90193.053.72–
lohoclanaflusodnE)56.7(66.67)58.9(33.3713.028.82–)97.8(48.77)91.01(28.4753.093.72–)65.5(48.08)88.01(89.4722.095.41–
edizonefamorhC)48.7(66.701)14.41(05.80113.064.42–)70.01(0.011)12.51(33.90104.048.55–)07.9(43.101)24.31(38.11193.043.33–
b-naflusodnE)42.11(43.87)60.51(05.8754.052.32–)44.21(43.08)38.41(55.9705.022.22–)25.01(0.28)20.31(27.1824.089.14–
noihtE)08.8(43.98)77.01(76.4953.083.5–)04.01(43.19)77.01(38.5924.022.4–)77.9(89.69)67.21(00.8993.030.74–
p,p¢-DDD)02.8(0.38)89.31(05.8933.086.01–)13.01(0.68)48.41(38.9914.091.91–)30.11(43.98)56.41(05.10144.057.11–
p,o¢-TDD)25.01(66.68)75.01(05.2924.053.4–)25.11(43.98)30.11(05.3964.013.3–)43.21(30.18)40.31(33.5905.043.91–
sohpozairT)44.11(43.49)91.41(71.6764.084.43–)00.01(66.69)12.41(05.7704.033.33–)42.9(65.98)28.11(76.1873.064.8–
MlyxalaneB)13.21(0.78)83.71(39.4794.082.91–)59.9(66.98)12.31(34.6704.066.71–)27.6(33.29)79.61(35.8772.095.01–
etaflus-naflusodnE)72.21(43.37)61.11(24.3894.052.52–)60.11(0.67)87.11(33.4844.034.42–)86.8(43.97)35.9(02.6853.006.2–
elozanociporP)16.11(43.87)45.11(33.30174.067.2–)53.9(66.08)76.11(05.40183.066.1–)49.7(43.48)28.9(38.60123.039.4–
p,p¢-TDD)03.8(43.78)72.51(33.60133.051.4–)00.8(0.19)24.51(76.70123.059.2–)25.7(43.49)66.11(05.31103.058.15–
elozanocubeT)74.9(00.09)78.41(00.5983.069.02–)05.21(66.29)11.31(71.6905.099.91–)92.9(66.59)41.41(38.9973.079.51–
etigraporP)23.01(48.97)15.3(05.8714.088.82–)45.21(69.08)00.5(76.9705.028.72–)42.11(06.19)83.3(00.2854.032.8–
naflusobraC)84.8(43.68)60.11(33.4943.013.32–)98.01(66.78)59.11(05.5944.063.22–)93.11(66.09)58.21(20.9964.003.02–
nirhtnefiB)72.7(00.57)72.5(89.9892.081.7–)77.9(61.77)38.4(04.1993.027.5–)67.5(05.08)32.8(23.4932.071.41–
sohpolinA)73.21(00.68)88.5(75.2805.091.13–)94.21(43.88)69.4(56.3805.092.03–)85.8(43.09)44.5(05.5843.033.52–
niuqazaneF)12.01(66.401)74.8(73.6814.031.22–)86.41(66.701)73.9(57.7893.088.02–)25.11(0.69)74.8(86.0964.063.32–
nefidarteT)71.11(43.97)61.21(00.6854.084.9–)33.21(0.18)62.31(78.6805.075.8–)70.9(66.18)31.21(20.1963.030.03–
enolasohP)45.8(43.86)58.11(87.1943.073.32–)36.9(43.07)99.21(58.2993.084.22–)28.4(0.37)51.01(09.4991.060.71–

of 10 and 50 mL capacity were used. Standard 1.8 mL dark
glass autosampler vials were used to contain final extracts.
(f) Homogenizer.—Polytron, PT-MR-3100 (Kinemetica
AG, Lucerne, Switzerland).
(g) Incubator shaker.—Model No. ZHWY-200D
(Zhicheng, China).
Reagents
(a) Solvents.—Residue analysis grade acetonitrile (ACN),
ethyl acetate (EA), and toluene were purchased from J.T.
Baker (Phillipsburg, NJ).
(b) Purified water.—Prepared using a Milli-Q water
purification system (Millipore Corp., Billerica, MA).
(c) Anhydrous magnesium sulfate (MgSO4), sodium
sulfate (Na2SO4), and sodium chloride (NaCl).—Analytical
reagent grade anhydrous MgSO4, Na2SO4, and NaCl were
purchased from Merck India Ltd (Mumbai, India). The
MgSO4 and Na2SO4 were heated in a muffle furnace at
400–450°C for 5 h before use and stored in desiccator.
(d) Pesticide standards.—Certified reference standards
were obtained from Sigma-Aldrich/Riedel-de Haën/Supelco
(St. Louis, MO). Stock solutions of the individual
pesticide standards were prepared by accurately
weighing 10 (± 0.01) mg of each pesticide (Table 1) in a
volumetric flask (certified A class) and dissolving in 10 mL
ethyl acetate. These stock solutions were stored in dark vials
in a refrigerator at 4°C. A 10 mg/L intermediate stock
standard mixture was prepared by mixing appropriate
quantities of the individual stock solutions and diluting
accordingly. A working standard mixture of 1.0 mg/L was
prepared by diluting the intermediate stock standard solution,
from which the calibration standards within the range
5–200 ng/mL were prepared by serial dilution with EA.
(e) Internal standard.—Triphenylphosphate (TPP) was
obtained from Sigma-Aldrich and used as an internal standard
(IS). A working IS concentration of 10 mg/mL in EA was
prepared and added to the test sample during sample
preparation. An appropriate dilution of this IS to 1 mg/mL with
EA was also prepared and used for the preparation of the
matrix-matched calibration standards.
(f) Analyte protectant.—Diethylene glycol (DEG; Merck,
Mumbai, India) was used as the analyte protectant, and its
working solution was prepared in EA (200 mg/mL).
(g) SPE sorbents.—These included primary-secondary
amine (PSA; Varian, Harbor City, CA; 40 mm particle size),
Bondesil C18 (ODS; Varian), graphitized carbon black (GCB;
United Chemical Technology, Bellefonte, PA), Florisil
(60–100 mesh; Acros, Geel, Belgium), Bond Elute amino
(Varian), and silica (60–120 mesh; Qualigens, Mumbai,
India).
(h) Tea samples.—Made tea was purchased from
Bio-garden of Makaibari Tea and Trading Co. (P) Ltd,
Kurseong, Darjeeling, India, and was used in fortification
experiments and as matrix blanks for matrix-matched
calibration standards.
(.2elbaTdeunitnoc)
edicitseP
lytubpofolahyC)20.21(0.07)96.31(33.8984.089.9–)12.21(66.17)37.51(76.9994.067.8–)10.01(4.47)14.41(71.20104.020.46–
zartimA)74.11(0.67)71.11(33.7864.004.61–)68.11(0.97)44.11(38.8884.079.41–)38.11(66.18)92.01(76.1984.086.71–
lnirhtolahyC-)52.01(66.39)17.5(33.1814.002.82–)52.01(0.69)98.6(24.2814.052.72–)50.11(0.101)30.7(24.5844.029.61–
anirhtemrepyC-)16.11(43.77)49.9(38.10174.031.4–)12.11(66.97)37.11(84.20154.015.3–)03.21(43.38)29.11(29.40194.059.41–
xorpnefotE)97.8(66.28)94.41(38.0953.037.31–)05.21(07.48)42.21(33.2905.013.21–)45.21(63.78)22.41(76.6905.031.11–
II+IetarelavneF)20.01(66.19)11.7(00.0904.072.51–)20.21(66.39)16.5(33.1984.010.41–)71.9(0.201)32.8(33.8873.063.73–
GetanilavulF-)12.9(66.09)70.8(00.5873.025.61–)14.01(66.68)53.01(00.6824.045.51–)10.8(8.88)83.71(05.2823.056.04–
elozanoconefiD)50.11(0.58)06.51(00.0944.048.9)57.9(0.78)25.41(71.1993.010.33)78.9(0.09)30.21(76.3904.004.32
nirhtemartleD)88.01(43.08)53.21(33.1844.008.62–)27.11(65.97)24.31(76.2874.006.52–)88.01(63.68)29.9(38.4844.071.36–
a
;)%,DSR(%,yrevoceRn.6=
b
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c
.tnemecnahnero)ngis”–“(noisserppuslangisdecudni-xirtamotsniatrep%,EM

Selection of Pesticides
We selected 67 compounds considering the pesticide use
pattern in Indian tea gardens, which also included persistent
organic pollutants like DDT analogs. The analytes belonged
to different chemical classes like carbamate, organochlorine
(OC), organophosphorus (OP), azole, synthetic pyrethroid,
strobilurin, cyclodiene, dinitroaniline, and nereistoxin. For
academic interest, we also included some acetanilide
herbicides. The details of the GC/MS parameters are
presented in Table 1.
Made Tea to Infusion Preparation
Made tea (5 g) was infused in 150 mL boiled water. After
3 min of brewing, the hot aqueous extract was filtered and
cooled. Tea infusion, spent leaves, and made tea were taken
for residue analysis.
Fortifications
In recovery studies, a calculated volume of the 1.0 mg/L
working standard mixture was added to each of the 1 g blank
(untreated) samples of made tea and spent leaves and 10 mL
tea infusion. The tube containing the fortified sample was
mixed on a vortex mixer for 30 s and left standing for 1 min to
allow even distribution of the pesticides and give time to
interact with the matrix.
d-SPE Cleanup
In the d-SPE cleanup approach, we compared the
following combinations of different sorbents: PSA, amino
(-NH2), Florisil, GCB, silica gel (Si), and ODS to obtain better
analyte recovery and less matrix interference from tea liquor,
spent leaf, and made tea. The combinations included (1) No
sorbent, (2) 25 mg PSA, (3) 25 mg PSA + 25 mg GCB,
(4) 25 mg PSA + 25 mg GCB + 25 mg Florisil, (5) 25 mg
PSA + 25 mg GCB + 25 mg NH2, (6) 25 mg PSA + 25 mg
GCB + 25 mg ODS, (7) 25 mg PSA + 25 mg GCB + 25 mg
Si, (8) 25 mg PSA + 15 mg GCB + 25 mg Florisil, (9) 25 mg
PSA + 20 mg GCB + 25 mg Florisil, (10) 25 mg
PSA + 40 mg GCB + 25 mg Florisil, and (13) 25 mg
PSA + 50 mg GCB + 25 mg Florisil. In addition to the above
combinations, 150 mg Na2SO4/mL extract was also used in
every case. The above cleanup experiments were done with
2 mL of organic phase extract. Toluene (0, 10, 20, and 30%)
was used with the organic phase, except for the first two
combinations.
Extraction and Cleanup Procedure for Made Tea,
Infusion, and Spent Leaves
Tea samples (made tea and spent leaves: 1 g) were taken in
50 mL FEP centrifuge tubes and mixed with 10 mL distilled
water, 100 mL of 10 mg/mL IS, 10 mL EA–cyclohexane
(9 + 1, v/v), and 1 g NaCl by mixing on a vortex mixer for 30 s
followed by blending for 1 min at 15 000 rpm in a Polytron
homogenizer. The homogenized samples were then
centrifuged at 3500 rpm for 5 min.
For the d-SPE, 1.6 mL supernatant and 0.4 mL toluene
were transferred into a 10 mL centrifuge tube prefilled with
25 mg each of PSA, GCB, and Florisil plus 300 mg Na2SO4.
The mixture was mixed on a vortex mixer for 30 s and
centrifuged at 6000 rpm for 10 min. For GC/MS analysis, a
1 mL aliquot was transferred from the supernatant to an
autosampler vial with 30 mL/mL diethylene glycol solution in
EA as an analyte protectant.
From the prepared tea infusion (described above), a 10 mL
aliquot (cooled to room temperature) was transferred to a
50 mL centrifuge tube. The pesticides were extracted with
10 mL EA–cyclohexane (9 + 1, v/v) and 1 g NaCl. The spent
leaves were taken after drying with filter paper, and residues
were extracted by a similar procedure as followed for made
tea. The cleanup procedure for tea infusion and spent leaves
was also similar to that of the made tea.
GC/MS Analysis
GC analysis was conducted on a capillary column (VF-5
MS, 30 m, 0.25 mm id, 0.25 mm film thickness; Varian,
Middelburg, The Netherlands) with the following conditions:
constant flow of helium at 1.3 mL/min; initial inlet
temperature of 75°C ramped to 280°C at 200°C/min after
a 20 s delay; and injection volume of 8 mL (large volume
injection) onto a Carbofrit plug in the liner with an open purge
valve (30:1 split ratio) for 18 s, closed until 3.5 min, and open
again (30:1) until the end of the run. The oven temperature
program included an initial temperature of 70°C (hold
for 2 min), ramped at 20°C/min to 180°C, ramped at 5°C/min
to 200°C with a hold for 3 min, ramped at 5°C/min to 220°C,
ramped at 7°C/min to 240°C, and finally ramped at 10°C/min
to 285°C with a hold for 3 min (total run time: 28.86 min). The
temperature of the transfer line, ion trap, and manifold were
set at 200, 230, and 60°C, respectively. Baseline offset of –5,
peak find with S/N of the quantifier ion of at least 3, and
peak width of 2 s were set as the peak processing parameters.
Minimum similarity match with regard to the National
Institute of Standards and Technology library spectra was
kept at 500 (reversed fit). Quantification was done on the basis
of the diagnostic ion (Table 1), and the peak assignments and
integration were automatically done through the software.
Preparation of Matrix-Matched Calibration
Standards
For calibration in GC/MS, six concentration levels
(5, 10, 20, 50, 100, and 200 ng/g) were prepared. The
matrix-matched calibration standards were prepared using a
sample:solvent ratio of 1:1. For calibration in fortification
experiments, the matrix-matched standards were prepared by
adding the appropriate volumes of the pesticide standards
mixture, IS, and analyte protectant solutions to each blank
extract.

seitniatrecnulabolgdnalaudividnI.3elbaTa
noisufniaetdna,aetedam,sevaeltnepsniedicitsephcaerof
edicitseP
U1U2U3U4U5UU2U1U2U3U4U5UU2U1U2U3U4U5UU2
otek-3narufobraC20.0810.0120.0520.0720.050.001.040.0110.0610.0320.0920.060.021.030.0610.0810.0210.0120.050.090.0
etarohP20.0120.0610.0020.0130.050.001.020.0620.0730.0030.0930.070.041.010.0090.0310.0610.0220.001.091.0
a-HCH20.0530.0340.0330.0140.080.061.010.0030.0230.0340.0940.080.061.050.0610.0810.0720.0130.070.041.0
etamyxorypneF20.0130.0150.0720.0620.070.051.020.0720.0520.0910.0820.050.011.040.0810.0410.0720.0330.060.031.0
b-HCH40.0140.0850.0330.0630.001.091.030.0320.0520.0630.0440.070.051.020.0020.0320.0420.0830.060.021.0
g-HCH60.0540.0940.0330.0530.001.002.040.0620.0030.0250.0860.001.002.080.0010.0910.0130.0830.001.091.0
nilarolhculF10.0740.0620.0040.0070.001.091.020.0050.0110.0320.0920.070.031.020.0030.0820.0630.0140.070.041.0
linolahtorolhC10.0150.0650.0060.0260.021.032.010.0120.0920.0230.0540.070.031.030.0240.0130.0150.0450.001.091.0
d-HCH20.0050.0130.0020.0220.070.041.030.0820.0620.0730.0840.080.061.020.0900.0410.0910.0720.040.080.0
edirolhcordyhpatraC20.0010.0610.0130.0430.050.011.030.0600.0210.0120.0720.050.090.030.0510.0420.0020.0720.050.011.0
sovrolhciD40.0820.0730.0820.0520.070.041.040.0900.0310.0720.0130.060.021.030.0240.0740.0230.0920.080.061.0
rolhcotecA10.0050.0120.0620.0130.070.041.020.0340.0060.0720.0130.090.071.020.0710.0520.0030.0730.060.021.0
rolhcalA10.0220.0420.0040.0340.070.041.010.0910.0820.0810.0320.050.090.020.0900.0310.0810.0720.040.080.0
lygraidaxO40.0140.0340.0050.0730.090.091.020.0430.0840.0920.0220.070.041.020.0110.0310.0920.0230.050.001.0
noihtalaM10.0720.0920.0020.0520.050.001.020.0090.0520.0810.0820.001.002.030.0420.0820.0410.0910.050.011.0
sofiryprolhC10.0620.0330.0130.0630.060.031.010.0630.0230.0620.0830.070.031.040.0430.0920.0220.0720.070.041.0
enoflusetarohP10.0010.0220.0020.0620.040.080.010.0510.0110.0030.0730.050.001.030.0610.0420.0230.0840.070.041.0
edixoflusetarohP30.0030.0320.0150.0070.001.002.020.0120.0620.0840.0240.070.051.050.0020.0420.0910.0120.070.031.0
nirdlA10.0920.0830.0040.0340.080.051.010.0400.0310.0800.0820.030.070.040.0810.0410.0610.0920.060.011.0
bracneboihT10.0620.0130.0030.0330.060.021.020.0630.0240.0720.0920.070.041.020.0350.0940.0610.0810.080.061.0
elozanocarteT20.0900.0410.0040.0640.070.031.020.0530.0510.0700.0110.040.090.030.0320.0720.0740.0940.080.061.0
lofociD10.0010.0120.0020.0220.040.080.020.0610.0020.0230.0830.060.021.040.0610.0810.0320.0720.060.021.0
nilahtemidneP10.0820.0930.0250.0060.090.091.010.0850.0570.0710.0910.001.002.020.0800.0410.0610.0920.040.080.0
linorpiF10.0130.0240.0260.0060.001.002.020.0810.0320.0530.0330.060.021.020.0210.0410.0320.0920.050.090.0
p,o¢EDD-10.0900.0710.0020.0420.040.080.010.0660.0950.0230.0430.001.002.020.0630.0140.0720.0920.070.041.0
rolhcatuB10.0130.0630.0320.0140.070.041.020.0720.0440.0410.0220.060.021.010.0140.0050.0810.0020.030.060.0
anaflusodnE-10.0710.0420.0520.0920.050.001.010.0730.0740.0430.0830.080.061.010.0630.0520.0720.0320.060.011.0
elozanocaxeH10.0240.0840.0420.0620.070.051.040.0010.0210.0020.0820.060.011.030.0440.0720.0810.0610.060.031.0
rolhcaliterP10.0320.0820.0020.0720.050.001.010.0510.0610.0910.0320.040.080.020.0410.0510.0920.0130.050.001.0
nibortsonimoteM20.0010.0120.0750.0760.090.091.030.0520.0720.0950.0840.090.081.040.0820.0720.0650.0750.001.091.0

(.3elbaTdeunitnoc)
edicitsePU1U2U3U4U5UU2U1U2U3U4U5UU2U1U2U3U4U5UU2
elozalcycirT50.0020.0220.0010.0210.060.021.050.0430.0740.0320.0030.090.071.070.0900.0910.0820.0830.090.071.0
p,p¢EDD-10.0800.0310.0910.0320.030.070.020.0090.0220.0510.0820.001.002.020.0530.0830.0710.0320.060.021.0
elozatubolcaP10.0910.0120.0610.0820.040.090.010.0110.0910.0320.0920.040.090.030.0310.0910.0620.0130.060.011.0
nirdleiD20.0110.0620.0910.0820.050.001.030.0720.0130.0710.0120.060.021.040.0110.0910.0130.0830.070.031.0
p,o¢DDD-10.0120.0520.0220.0420.050.090.010.0720.0920.0410.0710.050.090.020.0720.0130.0510.0110.050.001.0
nefroulfyxO10.0910.0720.0710.0020.040.090.020.0520.0510.0310.0810.040.080.010.0510.0910.0130.0350.070.031.0
nizeforpuB10.0020.0110.0810.0320.040.080.010.0820.0130.0710.0910.050.001.030.0520.0320.0410.0710.050.001.0
elozalisulF10.0320.0130.0520.0140.060.021.030.0720.0320.0710.0220.050.011.010.0900.0910.0720.0830.050.001.0
rypanefrolhC20.0620.0820.0520.0020.050.011.020.0620.0140.0910.0220.060.021.030.0610.0910.0220.0820.050.011.0
lohoclanaflusodnE10.0120.0420.0710.0410.040.080.040.0830.0840.0610.0910.080.051.020.0900.0310.0120.0620.040.080.0
edizonefamorhC50.0710.0420.0220.0330.070.041.060.0910.0320.0140.0440.090.081.040.0520.0430.0920.0720.070.041.0
bnaflusodnE-10.0720.0920.0610.0410.050.090.010.0410.0510.0720.0330.050.001.010.0420.0430.0840.0760.090.091.0
noihtE10.0130.0340.0240.0240.080.061.050.0900.0310.0320.0920.060.031.010.0610.0520.0230.0930.060.021.0
p,p¢DDD-10.0710.0320.0230.0330.060.011.010.0820.0130.0610.0910.050.001.020.0710.0420.0510.0020.040.090.0
p,o¢TDD-10.0220.0920.0320.0720.050.001.020.0110.0610.0920.0530.050.011.030.0210.0910.0140.0850.080.061.0
sohpozairT10.0060.0940.0340.0530.001.091.010.0720.0220.0710.0120.050.090.020.0900.0910.0130.0830.060.011.0
MlyxalaneB10.0320.0210.0420.0910.040.080.010.0420.0920.0810.0910.050.090.020.0410.0810.0820.0130.050.001.0
etaflusnaflusodnE20.0030.0430.0910.0610.060.011.020.0210.0810.0420.0820.050.090.030.0700.0110.0410.0320.040.080.0
elozanociporP20.0520.0820.0420.0620.060.011.030.0920.0020.0810.0720.060.011.050.0560.0050.0310.0910.001.002.0
p,p¢TDD-10.0220.0520.0420.0630.060.011.010.0110.0710.0220.0820.040.080.040.0030.0630.0910.0520.070.041.0
elozanocubeT20.0910.0420.0710.0220.050.090.020.0410.0020.0920.0430.050.011.070.0210.0610.0520.0920.080.061.0
etigraporP10.0230.0430.0320.0630.060.031.020.0620.0510.0110.0700.040.080.050.0510.0910.0130.0830.070.051.0
naflusobraC10.0530.0840.0030.0230.070.051.020.0900.0310.0320.0920.040.090.040.0640.0050.0710.0120.080.071.0
nirhtnefiB10.0420.0810.0510.0020.040.080.010.0750.0160.0510.0910.090.071.020.0110.0910.0130.0830.060.011.0
sohpolinA20.0320.0220.0420.0140.060.021.010.0150.0130.0110.0310.060.031.040.0820.0910.0630.0830.070.051.0
niuqazaneF10.0610.0910.0420.0920.050.090.010.0510.0610.0120.0720.040.080.010.0610.0910.0220.0830.050.001.0
nefidarteT10.0060.0270.0710.0020.001.002.020.0110.0810.0320.0830.050.011.010.0900.0310.0420.0820.040.080.0
enolasohP10.0220.0910.0120.0230.050.001.020.0520.0420.0710.0310.050.090.020.0610.0210.0910.0420.040.080.0
lytubpofolahyC10.0210.0610.0910.0220.040.070.020.0210.0910.0420.0820.050.001.020.0620.0920.0530.0430.070.031.0
zartimA20.0550.0850.0710.0120.090.071.020.0720.0120.0510.0810.050.090.020.0140.0940.0310.0910.070.041.0

Method Validation
The analytical method was validated using the single-
laboratory validation approach (20). The performance of the
method was evaluated considering the following different
validation parameters:
(a) Calibration range.—The calibration curves for all of
the compounds in pure solvent and matrix were obtained by
plotting the peak area against the concentration of the
corresponding calibration standards at six calibration levels
ranging between 1 and 200 ng/g.
(b) Sensitivity.—The LOD was determined by considering
an S/N of 3 with reference to the background noise obtained
from the blank sample, whereas the LOQ was determined by
considering an S/N of 10 using matrix-matched standards.
(c) Precision.—The precision in terms of repeatability
(two different analysts prepared six samples each on a single
day) and intermediate precision (two different analysts
prepared six samples each on 6 different days) were
determined separately for a standard concentration of 50 ng/g
of all of the analytes. The Horwitz ratio (HorRat) pertaining to
intralaboratory precision, which indicates the acceptability of
a method with respect to precision (21, 22), was calculated for
all of the pesticides in the following way:
HorRat = RSD/PRSD
where PRSD is the predicted RSD = 2C–0.15
and C is the
concentration expressed as a mass fraction (50 ng/g =
50 ´ 10–9
).
(d) Accuracy (recovery experiments).—Made tea
obtained from Bio-garden (which did not receive any
treatment with the test pesticides) was used as the blank. The
recovery experiments were carried out on fresh untreated
made tea, tea infusion, and spent leaves by fortifying the
samples in six replicates with a pesticide mixture separately at
two concentration levels, i.e., 50 and 100 ng/g, and the results
are reported in Table 2.
(e) Matrix effect.—The matrix effect (ME) was assessed by
using matrix-matched standards. The slope of the calibration
curve based on the matrix-matched standards of made tea, tea
infusion, and spent leaf was compared with the slope of the
pure solvent-based calibration curve. A higher slope of the
matrix calibration indicates matrix-induced signal
enhancement, whereas a lower slope represents signal
suppression. The ME wasevaluated by the following equation:
ME, % = (peak area of matrix standard – peak area of
solvent standard) ´ 100/peak area of solvent standard
In view of the above equation, the negative and positive
values of the ME signify matrix-induced suppression
and enhancement, respectively. Furthermore, in order to
minimize any errors in estimation, TPP (10 mg/mL) was used
as an IS.
(f) Measurement uncertainty.—Global uncertainty was
determined for all of the pesticides at the level of 50 ng/g
as per the statistical procedure of the EURACHEM/CITAC
(.3elbaTdeunitnoc)
edicitseP1U2U3U4U5UUU21U2U3U4U5UUU21U2U3U4U5UUU2
lnirhtolahyC-20.0710.0910.0510.0410.040.080.020.0610.0120.0210.0810.040.080.030.0620.0830.0120.0420.060.031.0
anirhtemrepyC-10.0630.0140.0620.0330.070.041.010.0710.0420.0410.0110.040.070.030.0920.0630.0810.0210.060.021.0
xorpnefotE20.0910.0610.0810.0420.040.090.020.0510.0910.0110.0310.040.070.020.0720.0030.0410.0910.050.001.0
II+IetarelavneF20.0220.0620.0920.0140.060.031.020.0710.0420.0820.0630.060.021.030.0310.0910.0130.0720.060.011.0
GetanilavulF-20.0910.0920.0520.0230.060.011.020.0820.0130.0710.0810.050.011.020.0410.0710.0810.0220.040.080.0
elozanoconefiD50.0320.0120.0330.0140.080.061.050.0920.0420.0810.0020.070.041.030.0800.0310.0120.0320.050.090.0
nirhtemartleD70.0410.0610.0420.0920.080.061.070.0620.0130.0210.0410.080.071.040.0610.0910.0310.0210.050.001.0
a
.g/gn05tadetaluclaC

Guide CG 4 (23) in the same way as reported by
Banerjee et al. (24). Five individual sources of uncertainty
were taken into account, viz., uncertainty associated with the
calibration curve (U1), day uncertainty associated with
precision (U2), analyst uncertainty associated with precision
(U3), day uncertainty associated with accuracy/bias (U4), and
analyst uncertainty associated with accuracy/bias (U5). The
global uncertainty (U) was calculated as:
U U U U U U= + + + +( )1
2
2
2
3
2
4
2
5
2 1 2
and was reported as expanded uncertainty, which is twice the
value of the global uncertainty. The uncertainty values for
each pesticide are reported as relative uncertainties in Table 3.
Results and Discussion
Selection of the Extraction Solvent
EA, ACN, and EA–cyclohexane in the ratio of 9 + 1,
8 + 2, 7 + 3, 6 + 4, and 1 + 1 (v/v) were evaluated for their
extraction efficiency. The results of the Student’s t-test
performed on the comparative recoveries obtained by using
these solvents showed that the recoveries of all pesticides
were statistically equal at the 95% confidence level (Figure 1).
In the case of EA, the recoveries of less-polar pesticides like
synthetic pyrethroids and carbamates were more than 70%
when quantified with matrix-matched standards. With ACN
extraction, the results were similar, with recoveries being
above 75%. But with EA–cyclohexane (9 + 1, v/v), the
recoveries of all the pesticides, including carbamates and
synthetic pyrethroids, were more than 80% with better
precision. Precision in terms of HorRat at the 50 ng/g level
were less than 0.5 for all pesticides (Table 2), indicating
satisfactory repeatability and ruggedness of the methodology.
Increase of the cyclohexane proportion in the extracting
solvent mixture (EA–cyclohexane; 8 + 2, 7 + 3, 6 + 4, and
1 + 1, v/v) did not result in any significant increase of the
recovery percentage. Relatively less recovery of
chlorothalonil was found irrespective of the extracting solvent
used, which is in accordance with the literature (25).
From this study, it is clear that EA–cyclohexane (9 + 1,
v/v) gave a higher recovery than any other solvent or solvent
mixtures tested for extraction of tea matrix.
Comparison of Shaking Versus Blending Versus
Mixing on a Vortex Mixer
The extractability of polar and nonpolar residues was
assessed through comparison of shaking versus blending
versus mixing on a vortex mixer to achieve the best initial
extraction step to be followed for made tea and spent leaf.
Most MRMs for pesticides in tea use a blender during
extraction (15, 26) but Gupta et al. (27) validated and
implemented a shaking procedure for tea. Our results revealed
that blending gave better recovery for most of the pesticides
compared to mixing on a vortex mixer and shaking-based
methods (Figure 2). Thus, we adopted a blending
homogenization procedure for extraction of residues from
made tea and spent leaves.
Comparison of Various Salts to Induce Phase
Separation
In developing our method, we used different sets of salt
combinations, e.g., NaCl, NaCl + MgSO4, and NaCl + Na2SO4,
with 10 mL EA–cyclohexane (9 + 1, v/v) in the initial extraction
step. In terms of the recovery of OC, OP, and other polar and
nonpolar pesticides, we did not achieve higher recovery by using
MgSO4 or Na2SO4 (Figure 3). But the amount of NaCl (1 g)
added to this system had a strong influence on the separation
between water and the organic phase.
Comparative Efficiency of Different SPE Sorbents
Tea matrix contains high amounts of polyphenols, methyl
xanthines such as caffeine, purines, and phenolic acids (28).
The main aim of the cleanup step was to remove these
coextractives as much as possible from the extract by using
different sorbents. The most commonly used sorbents include
Figure 1. Extraction capabilities of different solvent
systems; cyclohex = cyclohexane.
Figure 2. Extraction capabilities of blending over
shaking and mixing on a vortex blender for selected
pesticides.

weak ion exchangers (PSA or -NH2), GCB, strong anion
exchanger, and/or ODS SPE cartridges (29–33).
The d-SPE combinations No. 4 and 6 described in the
Experimental section gave statistically better results than
other combinations (Figure 4). But considering the high price
of ODS, Florisil is the best sorbent option in our opinion. So
the combination of PSA, GCB, and Florisil is the ideal
cleanup sorbent for removal of a variety of coextractives,
although, unfortunately, GCB retains structurally planar
pesticides such as HCH, phosalone, and chlorothalonil. To
investigate how much toluene is required to prevent recovery
loss on account of adsorption on GCB, the cleanup
experiment was repeated with standard solutions of 10, 20, or
30% toluene in EA. As apparent from Figure 5, even 10%
toluene dramatically improved the recovery. With 20%
toluene, the recovery of all the pesticides was higher than
70%, but use of 30% toluene did not show any significant
improvement in recovery percentage. So, it was concluded
that 20% was the optimum amount of toluene.
Analyte Protectant
To overcome the problem of the “matrix-induced signal
enhancement effect,” many laboratories use different types of
analyte protectants in pesticide analysis (34, 35). However,
DEG, 200 mg/mL in EA, was used in this studybecause it covers
theentirevolatilityrangeof thetargetpesticides.Theuseof DEG
as an analyte protectant dramatically reduced the errors caused
by matrix effects, as presented in Figure 6. The long-term effect
of DEG on the life of the liner/column and instrument sensitivity
needs to be examined, although it did not have any short-term
adverse effect.
Method Validation
All of the 67 pesticides could be analyzed by a single GC
run of 28.82 min at a 50 ng/mL or lower level. Linearity (r) of
the calibration curve, both for pure solvent-based as well as
matrix-matched standards, was >0.99 for most of the
compounds. LOQ values for all of the test pesticides (Table 1)
were below the maximum residue limit (MRL) values of the
respective compounds in/on tea as fixed by the EU (9). The
matrix-induced suppression in target signals was prominent
for a large number of pesticides, which possibly occurred as a
result of suppressions in the ionization process. Response
enhancement due to matrix effects was also observed for some
pesticides, viz., difenoconazole, butachlor, oxadiargyl,
chlorothalonil, and phorate. The slopes of the matrix-matched
calibration curves were significantly different compared to
pure solvent-based calibrations at the 95% level of statistical
confidence for each of the tea matrixes. An overall signal
suppression of 0.91–64% as well as signal enhancement
of 0.46–33% were observed irrespective of tea matrixes
(Table 2). Considering the variable matrix influences for
different compounds in mixtures, matrix-matched calibrations
were used for respective matrix-based quantification purposes
to avoid any over- or underestimation of residues. Our method
was quite satisfactory in terms of the RSD of less than 20%
(n = 6) for each compound.
Pesticide Recoveries and Repeatabilities
The results (Table 2) of recovery experiments at 50 and
100 ng/g with different tea matrixes show a satisfactory range
of 70–120%. The HorRat of all analytes calculated at the
50 ng/mL level of fortification was below 0.5 in spent leaf,
made tea, and tea infusion. Thus, the method provided
satisfactory levels of intralaboratory precision and accuracy.
Measurement Uncertainty Analyses
The total uncertainty was evaluated assuming that all the
contributions were independent of each other. A coverage
factor of 2 was chosen at a confidence level of 95% to evaluate
the expanded uncertainty at 50 ng/mL fortification (Table 3).
On the basis of expanded uncertainties, the test pesticides
could be classified into four groups: group I (up to 10%),
group II (10–15%), group III (15–20%), and group IV
(>20%). In the case of spent leaves, 17 pesticides could be
graded as group I, 32 as group II, 17 as group III, and one as
group IV. In spent leaf, higher uncertainty (>20%) was
recorded for chlorothalonil, which might be due to its basic
sensitivity. In the case of made tea and tea infusion, 20 and 13
pesticides were graded as group I, 33 and 41 as group II, and
14 and 13 as group III, respectively. In addition, most of the
pesticides (irrespective of matrixes) had low uncertainties
(£3%) associated with bias. It could, therefore, be concluded
Figure 3. Effect of different salts on the phase
separation.
Figure 4. Cleanup capabilities of different sorbents.

that the method optimized for sample preparation and analysis
is efficient enough and suitable for determination of pesticide
residues in these matrixes.
Economics of Analyses
The total cost of sample preparation (consumable inputs
only, excluding the instrument running cost, manpower, and
other overhead costs) was INR 150/sample, which is
equivalent to approximately $3/sample. Our estimate assumes
that a single laboratory chemist, on average, can process
around 20 samples in eight working hours performing the
activities starting from weighing the sample until it is ready to
inject for GC/MS analysis. The relative output increases to
above 30 samples/person/day when a group of chemists work
hand-in-hand, processing the samples in a chain mode.
Conclusions
The multiresidue GC/MS method developed and validated
could analyze 67 pesticides in tea samples with a GC run of
Figure 5. Effect of amount (%) of toluene in EA on the
recovery of pesticides adsorbed by GCB (12.5 mg/mL).
Figure 6. Effect of using analyte protectant (DEG). PP is the chromatographic plot preference.

28.82 min. The extraction process using EA–cyclohexane
(9 + 1, v/v) proved to be optimal for extracting multiclass
pesticides from tea samples. With d-SPE cleanup by
PSA + GCB + Florisil, high cleanup efficiency and low
matrix effects could be obtained, enabling use of this sensitive
and selective method for routine multiresidue analysis of
pesticides in tea matrixes with satisfactory recovery
(70–120%). The method is cost-effective and also offers a low
level of measurement uncertainty (£20%) for the test
compounds, indicating suitability for the requirements of
international standards.
Acknowledgments
We are grateful to Sanjay Dave, Director, APEDA,
Ministry of Commerce, Government of India, New Delhi, for
financial support. We are also thankful to Bidhan Krishi
Viswavidyalaya, West Bengal, India, for constant support
and inspiration.
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