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A multi-residue method for the analysis oforganophosphorus residues in cooked and polishedrice using accelerated solvent extraction anddispersive-solid phase extraction (D-SPE) technique anduncertainty measurementDoyeli Sanyal a , Anita Rani a & Samsul Alam aa Analytical Division , Institute of Pesticide Formulation Technology (IPFT) , Haryana, IndiaPublished online: 04 Sep 2009.

To cite this article: Doyeli Sanyal , Anita Rani & Samsul Alam (2009) A multi-residue method for the analysis oforganophosphorus residues in cooked and polished rice using accelerated solvent extraction and dispersive-solid phaseextraction (D-SPE) technique and uncertainty measurement, Journal of Environmental Science and Health, Part B: Pesticides,Food Contaminants, and Agricultural Wastes, 44:7, 706-716, DOI: 10.1080/03601230903163830

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Journal of Environmental Science and Health Part B (2009) 44, 706–716Copyright C© Taylor & Francis Group, LLCISSN: 0360-1234 (Print); 1532-4109 (Online)DOI: 10.1080/03601230903163830

A multi-residue method for the analysis oforganophosphorus residues in cooked and polished rice usingaccelerated solvent extraction and dispersive-solid phaseextraction (D-SPE) technique and uncertainty measurement

DOYELI SANYAL, ANITA RANI and SAMSUL ALAM

Analytical Division, Institute of Pesticide Formulation Technology (IPFT), Haryana, India

Quick, simple and efficient multi-residue analytical methods were developed and validated for the determination of organophospho-rous insecticides from polished and cooked rice. Polished rice was extracted using a simple, automated technique namely acceleratedsolvent extraction (ASE) using dichloromethane as the extraction solvent. Cooked rice was extracted with acetone and cleaned upusing dispersive-solid phase extraction (D-SPE) technique. The single step extraction method adopted for polished rice using acceler-ated solvent extractor provided satisfactory recovery for eight organophosphorus pesticides (OPPs) which ranged from 85.5–116.7%;90.0–110.3% and 93.5–118.8% at 1, 5 and 10 limit of quantification (LOQ) levels, respectively. The recovery of cooked rice was inthe range of 74–124%; from 75–100% and from 73–87% for 1, 5 and 10 level of fortification, respectively. The total uncertainty wasevaluated, taking four main independent sources viz., weighing, purity of the standard, GC calibration curve and repeatability underconsideration. The expanded uncertainty was found to be in the range of 5–20%.

Keywords: organophosphorus; multi-residue method; cooked rice; polished rice; GC-FTD; D-SPE; ASE; uncertainty measurement.

Introduction

Rice (Oryza sativa) is a staple food for a large part ofthe world’s human population, especially in tropical LatinAmerica, and East, South & Southeast Asia, making it thesecond-most consumed cereal grain.[1] India is the secondlargest producer of rice after China. Its consumption ishighest in China (187 million metric ton in 2007) followedby India (144 million metric ton).[2]

The crop is grown during Kharif season and thehigh temperature and humidity due to rainfall con-tribute to pest outbreaks which is a major challengefor crop protection specialists for developing sustain-able rice pest management techniques. Major rice pestsinclude brown plant hopper (Nilaparvata lugens Stal),army worms, green leaf hopper (Nephotettix nigropic-tus Stal), gall midge (Pachydiplosis oryzae Wood-Mason),rice bug, hispa, yellow stem borer (Tryporyza incertulasWalker), rats and the weed Echinochloa crusgali.[3] The Cen-tral Insecticide Borad/Registration Committee (CIB/RC)

Address correspondence to Doyeli Sanyal, Analytical Division,IPFT, Sector 20, Udyog Vihar, Gurgaon 122016, Haryana, India.E-mail: doyelisanyal@gmail.comReceived March 20, 2009.

responsible for registration of pesticide products inIndia has recommended the use of mostly organophospho-rus insecticides viz., phorate, monocrotophos, malathion,quinalphos, dichlorovos, phosphamidon, chlorpyrifos, fen-itrothion, phenthoate etc. for combating these insect pestsof rice.[4]

The paddy crop goes through various stages of process-ing such as cleaning, hulling, milling, and polishing beforeit is consumed after washing and steaming/boiling in wa-ter. To study the risk assessment and processing effect onthe fate of pesticides sprayed, it is important to have soundand effective analytical methods for analyzing them.

Very few research papers have been published on thedevelopment of multi–residue method for rice, either rawor cooked.[5−13] Earlier authors have reported the use oftraditional methods such as soxhlet,[5] shaker[6,7] and morerecently methods such as Matrix solid phase dispersion[8]

for extracting polished, unpolished and parboiled rice.Pengyan Liu et al.[7] developed a method in rice by GasChromatography-Mass Spectrometry Coupled with SolidPhase Extraction to simultaneously determine multiplepesticide residues in rice including organophosphorus,organochlorine, carbamate, and pyrethroid. Dorea andSobrinho[8] reported a multi-residue extraction methodbased on matrix solid-phase dispersion (MSPD) and

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Organophosphorus residues in rice 707

capillary gas chromatography–electron capture detectionfor quantification of two widely used organophospho-rus insecticides, malathion and parathion-methyl and oneorganochlorine pesticide, β-endosulfan in rice. Soon KilCho[9] used more recent technique viz., accelerated sol-vent extractor for extracting Diazinon, O-ethyl O-(4-nitrophenyl) phenyl phosphonothioate (EPN) and isopro-thiolane insecticides employing additional clean up stepwith florisil packed inside the extraction cell of ASE us-ing a solvent mixture of hexane + acetone at an elevatedtemperature of 100◦C.

In the case of cooked rice, limited research has been re-ported on the development of analytical method duringthe last decade.[11−13] Cogburn et al.[11] studied the fateof malathion and chlorpyrifos methyl in rough rice andmilling fractions before and after parboiling and cooking.The sample preparation involved shaking the rice with ace-tone in a wrist action shaker for 3 h and analyzing with gaschromatograph-flame photometric detector (GC-FPD) us-ing a glass 8% OV-101 column.

In this paper we report on the development of a singlestep ASE technique for the simultaneous extraction of nineorganophosphorus insecticides from polished rice. Accel-erated solvent extraction is a process that uses solvents atrelatively high pressure and temperatures at or above theboiling point.[14] The experimental parameters of the ex-traction are temperature, pressure, static time, cell size andthe solvent used.[15]

In the past decade, solid phase extraction (SPE) car-tridges filled with a variety of sorbents has been extensivelyused as an extraction and clean up tool. More recently, thedispersive-SPE (D-SPE) technique using bulk sorbents hasalso received wide application in residue analysis of pesti-cides. In this paper, we have employed the use of D-SPEtechnique for cleaning up of co-extractives from cookedrice.

The objective of the present study was to develop a simpleand efficient multi-residue method for the determinationof organophosphorus pesticides in polished and cookedrice. The developed multi–residue method was checked forvarious method performance characteristics such as accu-racy, sensitivity, linearity, limit of detection, ruggedness andmore importantly uncertainty of measurements as informa-tion on uncertainty has become very crucial for ensuringthe validity of the result.

Materials and methods

Chemicals

The certified analytical standards of all the nineorganophosphorous pesticides viz., DDVP (1,1-dichloro-2-dimethoxyphosphoryloxy-ethene, 99.7%), Monocro-tophos (Dimethyl (E)-1-methyl-2-(methylcarbamoyl)vinylphosphate, 98.5%), Phorate (O,O-diethyl S-ethylthiomethylphosphorodithioate, 98.5%), Phosphamidon (2-chloro-

2-diethylcarbamoyl-1-methylvinyl dimethyl phosphate,98.1%), Methyl Parathion (O,O-Diethyl-O-4-nitro-phenylthiophosphate, 96.5%), Fenitrothion (O,O-Dimethyl O-4-nitro-m-tolyl phosphorothioate, 100%),Malathion (2-(dimethoxyphosphinothioylthio) butane-dioic acid diethyl ester, 94.7%) Chlorpryrifos (O,O-diethylO-3,5,6-trichloro-2-pyridyl phosphorothioate, 100%) andQuinalphos (O,O-diethyl O-quinoxalin-2-yl phosphoroth-ioate, 99.2%), were procured from Accustandard Inc.(USA) in neat 10 mg (approx) weight. All the solvents andchemicals used in the study were of analytical reagent (AR)grade. Primary secondary amine (PSA, 40 µm, Bondesil)sorbent was purchased from Varian Inc., USA. Anhydroussodium sulfate purchased from Merck India Ltd. wassubjected to activation at 650◦C for 4 hours before use.Anhydrous magnesium sulfate was procured from Merck,Germany.

Instrumentation

A gas-chromatograph-flame thermionic detector (GC-FTD) (Flame Thermionic Detector, Shimadzu make, 2010model) equipped with autosampler (AOC-20i) and DB 1fused silica capillary column (J & W Scientific Co., 1%Phenylated methyl siloxane, 30 m length × 0.25 mm i.d. ×0.25 µm film thickness) column was used for quantificationof organophosphorus pesticides. Analysis was carried outusing an oven programming of initial temperature 100◦Cfor 1 min followed by a ramp rate of 40◦C/min up to afinal temperature of 200◦C followed by 3◦C/min ramp toa final temperature of 220◦C and finally a ramp rate of20◦C/min was given to reach a final temperature of 270◦Cwith a hold time of 2 minute. The injector temperaturewas set at 280◦C in splitless mode. The detector temper-ature was set at 300◦C at a current of 1.0 pA and makeup gas (Helium) flow at 29 mL/min. The carrier gas (He-lium) was set at a flow rate of 1 mL/min. The retentiontimes of DDVP, Monocrotophos, Phorate, Posphamidon,Methyl Parathion, Fenitrothion, Malathion, Chlorpyrifosand Quinalphos under this GC condition were 4.06, 6.20,6.64, 8.13, 8.40, 9.02, 9.24, 9.70 and 10.8 min, respectively.

Accelerated Solvent Extractor (ASE, Dionex, 200model) equipped with oven and 22 mL extraction cell and50 mL extraction vial was used for extraction of polishedrice. The ASE was operated at an ambient temperature at1500 psi pressure. The extraction was carried out with 3minute static time and the same repeated for 3 static cycles.The samples were flushed at 60% flush volume and purgedfor 100 seconds. The total extraction was completed within18 minutes and used only 33 mL.

Preparation of standard solution

The stock solutions of individual pesticide standards wereprepared by transferring the total amount of the pesti-cide contained in the vials (pre-weighed) into a volumet-ric flask (50 mL, Class A certified) and making up the

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volume with acetone (analytical reagent grade). The vialswere rinsed with acetone several times and the acetonewas transferred into the volumetric flask to ensure com-plete transfer. The vials were then air–dried and the emptyweight of the vial after complete transfer of the standardwas taken, up to four decimal in a precision analyticalbalance (Mettler Make). The difference in weight of thepre-weighed vial containing standard and the empty vialwas the actual weight of the pesticide standard transferred.The stock solutions thus constituted were stored at 20◦C.The concentrations of the individual stock solutions were268 µg mL–1 chlorpyrifos, 342.97 µg mL–1 DDVP, 404 µgmL–1 fenitrothion, 384.48 µg mL–1 malathion, 277.92 µgmL–1 methyl parathion, 281.71 µg mL–1 monocrotophos,274.91 µg mL–1 phorate, 386.51 µg mL–1 phosphamidonand 345.22 µg mL–1 quinalphos, respectively. An interme-diate stock standard mixture of 50 LOQ (Limit of Quantifi-cation) were prepared by mixing appropriate quantities ofthe individual stock solutions followed by requisite volumemake up. The concentration of each pesticide in 50 LOQstock standard mixture is enumerated in Table 1. Calibra-tion standards within 1-8 LOQ were prepared by serialdilution with acetone (Table 1).

Method validation

The quantification of pesticide was based one a five–pointcalibration graph obtained by plotting the detector re-sponse against concentration of the calibration standardswithin the range 1-8 LOQ (Table 1). Limit of Detection(LOD) and LOQ were calculated according to the peak-to-peak noise method.[16] The baseline of the unfortifiedblank at the analyte retention time window of interest wasmagnified to obtain the instrument response for the tallest(height) or the broadest (area) noise and converted theresponse into concentration (ppm or µg mL–1) from theknown concentration of the spiked extract. Limit of Detec-tion (LOD) was estimated by multiplying the response ofmethod noise level by approximately three and then con-verting the total response into concentration (µg mL–1).Limit of Quantification (LOQ) was estimated by multiply-ing the response of method noise level by approximately tenand then converting the total response into concentration(µg mL–1).

Matrix effect

The effect of matrix was assessed by preparing pesticidestandards in matrix blank extracted from untreated matrix.The matrix extracts were analyzed before spiking to confirmthe absence of the test pesticides in them. The effect ofsuppression or enhancement of peaks were assessed fromthe difference in the peak area of standard prepared inmatrix blank and the peak area of standard prepared insolvent blank.

Sample preparation and recovery studies

Polished riceRice was ground into a powder form in a grinder beforeuse. Representative sample (10 g) in triplicate were fortifiedat 1, 5 and 10 LOQ levels and extracted after 4 hours withdichloromethane in Accelerated Solvent Extractor (ASE)as per the conditions described above. The fortification at 1,5, and 10 LOQ were made by the addition of 0.4, 2 and 4 mLof 25 LOQ standard solution, respectively in 10 g of rice.Later 3.6 and 2 mL of acetone were added in 1 and 5 LOQsamples to maintain uniformity. The standard solution of25 LOQ was made by serial dilution of 50 LOQ stock.The concentration of each pesticide per gram of matrix in1, 5 and 10 LOQ spiking levels are enumerated in Table1. The extract was dried with anhydrous sodium sulphatefollowed by concentration using low volume concentrator(Turbo Vap LV, Caliper Life Sciences) using gentle nitrogenstream. The extracts were reconstituted with acetone to 3,12.5 and 25 mL in case of 1, 5, and 10 LOQ level samples,respectively. The final extract was filtered through syringeusing 0.2 µm membrane filter and analyzed in GC. Un-treated control in triplicate was also included in the studyin which 4 mL of acetone was added in place of pesticidestandard 4 hours prior extraction.

Cooked riceIn general, polished rice is sequentially subjected to rins-ing with water and steaming prior to consumption ascooked rice. In the present study, the polished rice wascooked in an open glass vessel using 150 mL of distilledwater with intermittent stirring for 20 minutes. The con-tents of the vessel were air cooled. The cooked rice (30g) in triplicate was fortified at 1, 5, and 10 LOQ levels(Table 1) in a similar manner as described above for pol-ished rice and extracted after 4 h. The contents of thevessel were transferred into a 500 mL stoppered Erlen-meyer flask with the help of 200 mL acetone. The flaskwas shaken at 320 rpm for 1 h in a rotary shaker. Theextracts were filtered through buchner filtration assemblyand concentrated to 10 mL using a rotary vacuum evap-orator (40◦C). The extracts were taken in a 500 mL sep-aratory funnel containing 100 mL of NaCl (5%, w/v) so-lution and partitioned using hexane + ethyl acetate (1:1,v/v, 100 + 50 + 50 mL). The extracts were dried usinganhydrous sodium sulfate and concentrated using rotaryvacuum evaporator (35◦C). The extracts were reconsti-tuted with acetone to 3, 12.5 and 25 mL in case of 1, 5,and 10 LOQ level samples, respectively. They were furthercleaned up by dispersive solid phase extraction techniqueusing primary secondary amine (PSA). Clean up was doneusing 3 mL of the extract. A total 25 mg/mL of PSAalong with 150 mg/mL of anhydrous magnesium sulfate(MgSO4) was added to the extract and centrifuged for 2min at 20,000 rpm. Finally, two microlitre of the aliquotfrom the centrifugation tube was injected in to the GC and

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Organophosphorus residues in rice 709

Tab

le1.

Con

cent

rati

onof

indi

vidu

alpe

stic

ides

atva

riou

sL

OQ

leve

lsin

polis

hed

and

cook

edri

ce.

Pol

ishe

dri

ce(µ

gm

L−1

)C

ooke

dri

ce(µ

gm

L−1

)

Pest

icid

e1

LO

Q2

LO

Q4

LO

Q5

LO

Q6

LO

Q8

LO

Q10

LO

Q50

LO

Q1

LO

Q2

LO

Q4

LO

Q5

LO

Q6

LO

Q8

LO

Q10

LO

Q50

LO

Q

DD

VP

0.07

80.

155

0.31

00.

388

0.46

50.

620

0.77

63.

878

0.01

00.

021

0.04

10.

052

0.06

20.

083

0.10

30.

517

Mon

ocro

toph

os0.

013

0.02

50.

050

0.06

30.

075

0.10

10.

126

0.62

80.

011

0.02

30.

046

0.05

70.

069

0.09

20.

115

0.57

3P

hora

te0.

009

0.01

70.

034

0.04

30.

052

0.06

90.

086

0.43

10.

019

0.03

70.

074

0.09

30.

111

0.14

90.

186

0.92

8Po

spha

mid

on0.

018

0.03

50.

070

0.08

80.

105

0.14

00.

175

0.87

60.

011

0.02

10.

042

0.05

30.

063

0.08

40.

105

0.52

6M

ethy

lPar

athi

on0.

010

0.02

10.

042

0.05

20.

063

0.08

40.

105

0.52

30.

003

0.00

60.

013

0.01

60.

019

0.02

50.

032

0.15

9F

enit

roth

ion

0.01

00.

021

0.04

20.

052

0.06

30.

084

0.10

50.

523

0.01

00.

019

0.03

80.

048

0.05

80.

077

0.09

60.

480

Mal

athi

on0.

012

0.02

50.

050

0.06

20.

074

0.09

90.

124

0.62

00.

016

0.03

10.

063

0.07

90.

094

0.12

60.

157

0.78

6C

hlor

pyri

fos

0.02

40.

047

0.09

40.

118

0.14

10.

188

0.23

51.

176

0.00

30.

005

0.01

10.

013

0.01

60.

021

0.02

60.

132

Qui

nalp

hos

0.00

20.

003

0.00

60.

008

0.00

90.

012

0.01

50.

075

0.00

30.

007

0.01

40.

017

0.02

10.

027

0.03

40.

171

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710 Sanyal et al.

analyzed. Untreated control in triplicate was also includedin the study in which 4 mL of acetone was added in placeof pesticide standard 4 hours prior extraction.

Determination of uncertainties

Combined uncertainty in estimation was determined for allthe pesticides at 5 LOQ level as per the statistical procedureof the EURACHEM/CITAC Guide CG 4.[17] Four indi-vidual sources of uncertainty were taken into account asdescribed below:

Uncertainty sources associated with GC calibrationAttention was paid to weighing, purity of the analyticalstandards and gas chromatograph (GC) calibration.

Uncertainty of purity of analytical standardsAs the uncertainty of standard purity declared in the sup-plier’s certificate was given without any confidence level,rectangular distribution was assumed for calculating stan-dard uncertainty (Equation 1).

SU1 = (u(x)/√

3) (1)

where u (x) represents the uncertainty value given in thecertificate for purity of standard.

Relative standard uncertainty (U1) was derived accord-ing to the equation (2).

U1 = (SU1 × 100)/% purity (2)

Uncertainty of weighingThe relative standard uncertainty due to weighing was cal-culated using normal distribution given by Equation 3.

U2 = (0.0001/2)/Wi (3)

where Wi is the weight of the pesticide standard weighedusing precision analytical balance, 0.0001 is the value of un-certainty of the balance at 95% confidence level as reportedin the certificate. The uncertainty of the balance has beendivided by 2 taking normal distribution into consideration.

Uncertainty of calibration linearityNon-linearity of the calibration curve can be also consid-ered as another source of uncertainty. Uncertainty asso-ciated with the calibration curve (U3) was calculated at 5LOQ level according to the Equation 4.

U3 =[

sb1

] [{1p

}+

{1n

}+

{(c0 − c′)2

sex

}]1/2

(4)

where s is the standard deviation of the residuals of thecalibration curve, b1 is the slope of the calibration curve,p is the number of measurements of the unknown, n isthe number of points used to form the calibration curve,c0 is the calculated concentration of the analyte from the

calibration curve, ′c is the arithmetic mean of the concen-trations of the standards used to make the calibration curveand sxx is calculated as given in Equation 5.

sxx = σ (cj − c′)2 (5)

where j =1, 2, ..., n. cj is the concentration of each calibra-tion standard used to build up the calibration curve.

Uncertainty associated with precisionIn the present study, the random errors of extraction, clean-up and GC analyses steps were approximated by standarddeviations which were calculated from repeated determina-tions of analytes expressed as repeatabilities. The precisionwas calculated according to the Equation 6.

U4 = s/(√

n ∗ x) (6)

where s is the standard deviation of the results obtainedfrom the recovery study, n is the number of assays and x isthe mean value of the concentration recovered.

The combined uncertainty (U) was calculated as U =x[(U12 + U22 + U32 + U42)1/2] and reported as expandeduncertainty (2U) which is twice the value of the combineduncertainty at 95% confidence level.

Results and discussion

Pesticide selection

Major pest infestation in paddy is due to insects such asstem borer, brown plant hopper, leaf folder, rice hispa,gall midge etc. in India. Out of the total pesticides rec-ommended in India for use against insects in paddy,the majority are organophosphorus insecticides. The nineorganophosphorus pesticides (OPPs) chosen for the studyare used widely in India.

Validation of the method

Linearity of calibration curve, LOD and LOQThe nine OPPs were resolved in an 11–minute run time. Thecorrelation coefficient (R2) was found to be ≥0.99 for eachpesticide in each matrix (Fig. 1). The pesticides presented alinear behavior in the standard concentration range of 1-8LOQ. LOD and LOQ were estimated as the lowest con-centration of pesticide injected that yielded a Signal/Noiseratio of 3 (LOD) and 10 (LOQ), respectively. The LOD andLOQ for the test pesticides are presented in Table 2. TheLOD and LOQ were in the range of 0.5 to 7.1 ng mL−1

and 1.5 to 23.5 ng mL−1 in case of polished rice with anexception of DDVP (LOD 23.3 and LOQ 77.6 ng mL−1),respectively. In case of cooked rice, LOD and LOQ were inthe range of 0.8 to 5.6 ng mL−1 and 2.6 to 18.6 ng mL−1,respectively.

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Organophosphorus residues in rice 711

0

100000

200000

300000

0.0000 1.0000 2.0000 3.0000 4.0000 5.0000 6.0000 7.0000 8.0000

LOQ

AR

EA

DDVP Monocrotophos Chlorpyrifos Methyl Parathion Fenitrothion

Phorate Phosphamidon Malathion Quinalphos

Fig. 1. Calibration curve of nine organophosphorus pesticides (refer to Table 1 for concentration of each pesticide).

Matrix effect

Enhancement in response due to matrix effect was promi-nent for all the test pesticides except monocrotophos inpolished rice. The enhancement ranged from 12 ± 1.6(quinalphos) to 22.5 ± 0.04% (DDVP). The signal suppres-sion for monocrotophos was as high as 49 ± 0.2%. How-ever, the signal enhancement in cooked rice was quite highin comparison to polished rice except for monocrotophosand phorate where about 55 and 22% suppression in GCresponse was observed, respectively. This variable matrixinfluence clearly implies that matrix matched calibrationstandards should be commodity specific for quantificationto avoid any over- or under estimation of residue levels.Representative chromatograms of polished and cooked riceare presented in Figure 2.

Table 2. Linearity, LOD and LOQ levels of organophosphoruspesticides in polished and cooked rice.

Polished rice Cooked rice(ng mL−1) (ng mL−1)

Peak no. Pesticide R2 LOD LOQ LOD LOQ

1. DDVP 0.997 23.3 77.6 3.1 10.32. Monocrotophos 0.998 3.8 12.6 3.4 11.53. Phorate 0.999 2.6 8.6 5.6 18.64. Posphamidon 0.999 5.3 17.5 3.2 10.55. Methyl Parathion 0.997 3.1 10.5 1.0 3.26. Fenitrothion 0.998 3.1 10.5 2.9 9.67. Malathion 0.997 3.7 12.4 4.7 15.78. Chlorpyrifos 0.997 7.1 23.5 0.8 2.69. Quinalphos 0.997 0.5 1.5 1.0 3.4

Recovery and precisionThe single step extraction method adopted for polished riceusing accelerated solvent extractor provided satisfactoryrecovery for eight organophosphorus pesticides (OPPs)(Table 3 and Fig. 3) which ranged from 85.5% (phos-phamidon) to 116.7% (monocrotophos); 90.0% (Phorate)to 110.3% (monocrotophos) and 93.5% (Chlorpyrifos) to118.8% (monocrotophos) at 1, 5 and 10 LOQ levels, respec-tively. However, recovery of DDVP was found to be belowthe acceptable range of 70–120%. The use of acceleratedsolvent extractor for rice matrix proved to be very con-venient. Extraction time and solvent volume was reducedconsiderably. Moreover, the extracts were clean enough tobe injected directly into the GC without any further cleanup. Earlier authors have reported the use of traditionalmethods such as soxhlet[5], shaker[6,7] and more recentlymethods such as Matrix solid phase dispersion[8] for ex-tracting polished, unpolished and parboiled rice. Howeverthere has been no report on the use of pressurized sol-vent extractor for extraction of rice till date except one bySoon Kil Cho et al.[9] Cho used accelerated solvent extrac-tor for extracting Diazinon and EPN insecticides employ-ing the additional clean up step using sorbent like florisilpacked inside the extraction cell of ASE using a solventmixture of hexane + acetone at an elevated temperature of100◦C.

Use of PSA for clean up considerably reduced thenoise level in cooked rice matrix blank when comparedto the samples either processed without Dispersive SPE orcleaned up with florisil. The recovery (Table 4 and Fig. 4)was in the range of 74 (monocrotophos) to 124% (DDVP);from 75 (monocrotophos) to 100% (chlorpyrifos) and from73 (DDVP) to 87% (fenitrothion) for 1, 5 and 10 level offortification, respectively.

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(a) Polished rice matrix blank (a) Cooked rice matrix blank

(b) 5 LOQ std in matrix blank (b) 5 LOQ std in matrix blank

(c) 5 LOQ recovery in polished rice

(c) 5 LOQ recovery in cooked rice

Fig. 2. Representative GC-NPD chromatograms of pesticides for (a) blank matrix (b) matrix matched standards at 5 LOQ level (c)recovery of pesticide in matrix fortified at 5 LOQ level. See Table 1 for the name of the labeled peaks (refer to Table 1 for concentrationof each pesticide).

In case of DDVP (dichlorovos), the low recovery in pol-ished rice may have been connected with the use of an evap-oration step prior to clean up and GC injection as DDVP isa volatile pesticide and is susceptible to volatilization lossesduring solvent evaporation.

The mean recoveries at three spiking levels viz., (a)>0.001 ≤0.01 (b) >0.01 ≤0.1 and (c) >0.1 ≤1 mg/Kg

for polished rice matrix was 95.6, 93 and 85.7%, respec-tively and were within Codex acceptable range.[18] The me-dian repeatability calculated as CV (%) was 5.6, 7.6 and8.0% for the aforementioned three spiking levels, respec-tively and was well below the Codex acceptable range.Similarly, in the case of cooked rice the mean recoverywas 87.3, 86.2 and 84.4% and median repeatability was

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Organophosphorus residues in rice 713

Table 3. Recovery (%) of the organophosphorus pesticides from polished rice at three fortification levels.

1 LOQ 5 LOQ 10 LOQ

Pesticide Mean recovery % CV∗ Mean recovery % CV∗ Mean recovery % CV∗

DDVP 43.9 13.1 44.2 30.6 35.6 8.95Monocrotophos 116.7 5.1 110.3 18.4 118.8 13.6Phorate 87.3 5.7 90.9 11.2 90.4 6.4Posphamidon 85.5 1.7 98.6 6.3 94.5 5.3Methyl Parathion 97.9 5.19 94.0 9.2 96.1 0.08Fenitrothion 87.9 4.2 100.7 7.7 97.8 2.1Malathion 88.9 5.44 95.7 7.6 96.2 2.0Chlorpyrifos 96.7 1.4 94.1 6.3 93.5 2.3Quinalphos 97.7 3.7 107.1 9.0 96.3 5.0

∗% CV = Relative standard deviation (RSD) = (standard deviation/average recovery) × 100.

8.8, 8.4 and 6.6%, respectively for the three spiking levels(Table 5).

Measurement of uncertainties

In the present study, the total uncertainty was evaluatedtaking four main independent uncertainties arising out ofweighing, purity of the standard, GC calibration curve andrepeatability under consideration.

The expanded uncertainty of the pesticides varied be-tween 8.7 to 21% except DDVP in the case of polished rice(Table 6). In cooked rice matrix, the uncertainty rangedbetween 6.6 to 21%. It can therefore be assumed that themulti–residue method adopted for the pesticides is suitableand efficient in determining these pesticides from the ma-trices discussed. The relatively high value of uncertaintyof DDVP is in conformity with its poor recovery from pol-ished rice showing that the method exhibited relatively poorperformance for this compound.

The contribution of individual uncertainties to the to-tal uncertainty values may be evaluated from Table 6.The uncertainty due to repeatability represents the most

important source of the total uncertainty (72%) as cal-culated for polished rice. This was followed by uncer-tainty due to purity of the pesticide standard which con-tributed 24%. Uncertainty of GC calibration proved tobe an insignificant component (0.01%) of the total uncer-tainty. Cuadros-Rodrıguez et al.[19] applied the “bottom-up” approach in estimation of uncertainty associated withdetermination of organophosphorus and organochlorinepesticides in cucumber. Repeatability of determination ofanalytes in spiked samples and also uncertainty associatedwith the preparation of the calibration standard solutions(weighing, diluting) were identified as the most significantsources of combined uncertainty. Radim Stepan et al.[20]

also using the “bottom-up” approach, showed that uncer-tainty of extraction (which comprised of two components:(i) repeatability of extraction and (ii) uncertainty of extrac-tion recovery) was shown to represent the main source ofcombined standard uncertainty On the other hand, uncer-tainties associated with the GC calibration (uncertaintiesof weighing and diluting standards, uncertainties of purityof standards) were not so important. Most of them did notexceed 2%.

Fig. 3. Recovery (%) of the pesticides from polished rice with error bars representing the standard deviation (refer to Table 1 forconcentration of each pesticide).

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Table 4. Recovery (%) of the organophosphorus pesticides from cooked rice at three fortification levels.

1 LOQ 5 LOQ 10 LOQ

Pesticide Mean recovery % CV∗ Mean recovery % CV∗ Mean recovery % CV∗

DDVP 124.6 17.3 76.3 6.1 73.6 3.8Monocrotophos 74.7 1.4 75.7 9.5 85.1 10.1Phorate 81.2 4.6 80.6 4.6 84.1 9.1Posphamidon 83.7 3.8 88.6 4.1 87.3 9.5Methyl Parathion 83.7 14.4 85.3 18.0 81.2 1.4Fenitrothion 82.5 8.4 83.7 4.1 87.5 4.2Malathion 81.9 11.3 80.7 9.6 82.0 8.1Chlorpyrifos 94.3 9.0 100.3 5.3 86.6 11.4Quinalphos 97.9 8.7 86.1 7.9 83.5 8.2

∗% CV = Relative standard deviation (RSD) = (Standard deviation/Average recovery) × 100.

Table 5. Overview of method validation characteristics for different matrices.

Accuracy Precision

Spiking level Mean Codex acceptable Repeatability of Codex acceptableMatrix (mg/Kg) recovery (%) range recoveries CVa (%) range

Polished rice >0.001 ≤0.01 95.6 60–120 5.6 30>0.01 ≤0.1 93 70–120 7.6 20>0.1 ≤1 85.7 70–110 8.0 15

Cooked rice >0.001 ≤0.01 87.3 60–120 8.8 30>0.01 ≤0.1 86.2 70–120 8.4 20>0.1 ≤1 84.4 70–110 6.6 15

aCoefficient of variation for the analysis.

Table 6. Results of individual and combined uncertainties for each pesticide calculated at 5 LOQ level (refer to Table 1 forconcentration of each pesticide).

Standard Pesticide Calibration Combined Expandedpurity Weighing curve repeatability Uncertainty uncertainty

S. no. Pesticide U1 U2 U3 U4 U 2U

Polished Rice1. DDVP 0.023 0.003 6E-06 0.177 0.0306 0.06122. Monocrotophos 0.023 0.003 6E-07 0.106 0.0076 0.01513. Phorate 0.024 0.004 5E-06 0.065 0.0027 0.00544. Posphamidon 0.024 0.003 1E-05 0.037 0.0038 0.00765. Methyl Parathion 0.024 0.003 5E-06 0.053 0.0029 0.00586. Fenitrothion 0.023 0.002 1E-05 0.045 0.0027 0.00537. Malathion 0.024 0.002 1E-05 0.044 0.0030 0.00608. Chlorpyrifos 0.023 0.004 3E-06 0.037 0.0048 0.00979. Quinalphos 0.023 0.003 2E-05 0.052 0.0005 0.0009

Cooked Rice1. DDVP 0.023 0.003 – 0.035 0.0125 0.02512. Monocrotophos 0.023 0.003 – 0.055 0.0028 0.00573. Phorate 0.024 0.004 – 0.027 0.0012 0.00254. Posphamidon 0.024 0.003 – 0.022 0.0025 0.00505. Methyl Parathion 0.024 0.003 – 0.104 0.0047 0.00956. Fenitrothion 0.023 0.002 – 0.023 0.0014 0.00297. Malathion 0.024 0.002 – 0.055 0.0030 0.00618. Chlorpyrifos 0.023 0.004 – 0.031 0.0046 0.00919. Quinalphos 0.023 0.003 – 0.046 0.0003 0.0007

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Organophosphorus residues in rice 715

Fig. 4. Recovery (%) of the pesticides from cooked rice with error bars representing the standard deviation (refer to Table 1 forconcentration of each pesticide).

Due to negligible contribution of uncertainty of GC cal-ibration, this source was ignored in the subsequent calcu-lations of uncertainty in the cooked rice matrix.

Conclusion

The multi–residue method developed for quantification oforganophosphorus pesticides from polished and cookedrice using modern extraction technique viz., ASE and cleanup technique viz., D-SPE proved to be efficient and cost–effective as it reduced the number of steps in sample prepa-ration. The recovery of nine OPPs were within the Codexacceptable range of 70–120% except DDVP in the case ofpolished rice. The expanded uncertainty was well below20% for most of the pesticides indicating efficiency of themulti–residue method.

Acknowledgment

The authors are thankful to Director, IPFT for providingthe necessary facilities required to carry out the presentwork.

References

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