Preliminary Spiked Recovery Test on Multi Residue Analyses of Organochlorine and Organophosphorus Pesticides KesinyPhomkeona, VanpaseuthPhouthavong, Kazuyuki Tsuzuki Department of Chemistry, Faculty of Science, National University of Laos, Dong Dok. Department of Life Science, Toyohashi University of Technology, Toyohashi, Japan.

ABSTRACT This work presents a preliminary test of a suitable extraction and clean up method for multi residue analyses of three organochlorine pesticides (2,4’-DDT, 2,4’-DDE and 2,4’DDD) and three organophosphorus pesticides (malathion, methyl parathion and ethyl parathion) followed by gas chromatography-electron capture detection (GC-ECD). A blank apple sample was pesticide added and undergone a single extraction by acetone/water solvent. Silica gel and Florisil were tested for the appropriate clean up column after the first extraction. The results indicate that silica gel was more suitable for cleaning up purpose and mixture of hexane and ethyl acetate was also found to be the most efficient eluting solvent of the silica column. In addition, all six studied pesticides can be separated within a single run by GC-ECD. Keywords:Multi residue analyses, Organochlorine pesticides, Organophosphorus pesticide, Gas Chromatography

1.

Introduction

In recent years, it is concerned about pesticide residue in foods in which it produces bad effect to human health. In Laos, the same situation is being encountered. Due to dependence on many imported foods, establishment of analysis is quite demanded. Especially, multi analyses method which can detect many pesticides in a single run is getting much attention because of its convenience, less time and chemical consuming. Recovery test is a very common parameter to check the accuracy of a proposed analysis method. The suitable method for multi analyses must enhance percentage recovery of each studied pesticide within a single analysis process. In this proposed work, percentage recoveries of three species of each organochlorinepesticides (2,4’-DDT, 2,4’-DDE, and 2,4’-DDD) and organophosphorus pesticide (malathion, methyparathion, and ethylparathion) were tested on a single extraction and single cleaning-up columnfollowed by gas chromatographic separation with electron capture detection. All procedures were modified from those of Food and Agricultural Materials Inspection Center (FAMIC, in Kobe). A. Cl

CHCl2

Cl

Cl

2,4'-DDD

Cl

Cl

Cl

2,4'-DDE

276

CCl3

Cl

Cl

2,4'-DDT

B. O O 2N O

S

EtO

S

P(OMe)2

EtO

P(OMe)2

S

O 2N

S O

P(OEt)2

O

Methylparathion

Malathion

Ethylparathion

Figure1. Chemical structures of the studied pesticides: Aorganochlorine pesticides, B organophosphorus pesticides

2. 2.1

Experiment Chemicals

Organochlorine pesticides standard (in cyclohexane) and organophosphorus pesticides standard (in acetonitrile) were of 10 ng/μL concentration (Dr. Ehrenstorfer GmbH). Two types of solid sorbents were used: silica gel 60 (0.063-0.200 mm), and Florisil®30-60 mesh (Aldrish). Sodium sulfate anhydrous, dried in 130℃oven over night before each use.Saturated sodium chloride solution prepared by recrystallization of household-use grade NaCl in water, use the supernatant. All organic solvents were of analytical reagent grade: acetone, n-hexane, cyclohexane, ethyl acetate, dichloromethane and acetonitrile (RCI LabScan). 2.2

Preparation of Silica gel and Florisil column

A 1.8×40㎝glass column with Teflon cock was filled with 20-cm dry silica gel or Florisiland2-cm sodium sulfate anhydrous on the top. Before using, the column was washed by 10 mL hexane contains 30 % of acetone and 15 mL hexane, respectively. 2.3

Instrumentation

A Gas chromatograph(Varian 3300) equipped with an electron capture detector (ECD) was employed for pesticide separation. Gaseous nitrogen was used as a carrier gas (2 mL/min) and also a make-up gas (28 mL/min). Splitless injection mode was used with 1µLinjection volume. Operational temperature of each part was: injector 250 ℃, detector 300 ℃, and column 60 ℃(2 min)→(20 ℃/min)→160 ℃(1 min)→(5 ℃/min)→230 ℃(10 min).

2.4

Extraction of pesticides

The sample was prepared from apples sold in alocal market. 10 g of the blended sample was added target pesticide 6 species (each 100 µL) and thoroughly mixed. After 1 hour, the paste was added silica gel 2 g and 20 mL acetone contains 30% water. Then 5 minutes homogenized, and vacuum filtration. Residual thing recovered from filter was repeated homogenizing 5 minutes with another 20 mL and 10 mL acetone contains 30 % water, respectively. All extract solutions were combined to become 50 mL solution. The 50 mL solution was transfered to a 500-mL separatory funnel and added 100 mL pure water, 50 mL saturated NaCl aqueous solution and 50 mL dichloromethane. After 3 times of extraction, the solution from organic layer became 150 mL and was dehydrated by using 20 g sodium 277

sulfate anhydrous.Then organic solvent was removed completely by rotary evaporator and redissolved in 1 mL hexane contains 30 % acetone. 2.5

Clean up a)

Method 1.

The 1 mL solution was fractionated in a Florisil column by four eluting solvents: 20 mL of n-hexane and 20, 80, 300 mL of hexane containing 30 % acetone, and named as FRACTION 1, 2, 3, 4, respectively. Solvents in those four fractions was then evaporated completely and redissolved in 1 mL cyclohexane for FRACTION 1 and in 1 mL acetonitrile for FRACTION 2, 3, 4 before GC-ECD analyses. b)

Method 2.

The 1 mL solution was fractionated in a Florisil column by a single eluting solvent: 500 mL of n-hexane containing 30 % acetone. The solvent was then evaporated completely and redissolved in 1 mL acetonitrilebefore GC-ECD analyses. c)

Method 3.

The 1 mL solution was fractionated in a silica gel column by four eluting solvents: 50 mL of n-hexane containing 30, 50, 70 and 100% ethyl acetate. Solvents in those four fractions was then evaporated completely and redissolved in 1 mL acetonitrile before GCECD analyses. 3.

Result and Discussion

Percentage recoveries of each pesticide added to the blank sample were considered to be the indication of suitable method for multi residue analyses of the studied pesticides. Percentage recoveries were calculated by the following formula: %𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑦 =

𝑃𝐴𝑓𝑜𝑢𝑛𝑑 × 100 𝑃𝐴𝑎𝑑𝑑𝑒𝑑

Where PAadded is peak area of standard pesticide added to the sample before extraction PAfound is peak area of standard pesticide found in the sample after extraction and clean up 3.1

Method 1

Three organochlorine pesticides were found in each fraction (see Table 1.). Almost 100% of 2,4’-DDD was recovered in FRACTION 3, while the other two organochlorines (2,4’-DDT and 2,4’-DDE) were around half amount recovered. Recoveries of FRACTION 4 indicated that increasing eluting solution volume to 300 mL could recover only 53.49 and 59.28% of 2,4’-DDE and 2.4’-DDT, respectively. On the other hand, all studied organophosphorus pesticides (malathion, methyl parathion and ethyl parathion) were found in FRACTION 3 and there was no recovery in the last fraction. However, the recovery of each organophosphorus was of comparable to that of organochlorine. It was also revealed that all six pesticides were found only in FRACTION 3.

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Table 1. %Recovery of each studied pesticide obtained by each fractionated eluting solvent on Florisil column

Pesticides

FRACTION1: 20mL hexane

2,4’-DDT 2,4’-DDE 2,4’-DDD Malation Methyl ND parathion Ethyl ND paration ND = Not detectable

15.44 12.75 56.29 39.40

%Recovery in each fraction FRACTION2: FRACTION3: FRACTION4: 20mL 30% 80mL 30% 300mL 30% acetone in acetone acetone in hexane hexane in hexane 25.74 10.62 7.48 23.27 9.31 8.16 21.63 23.19 ND 14.60 14.97 ND ND 17.00

Total

59.28 53.49 101.11 68.97

66.27 ND

62.35

39.31 ND

56.31

3.2

Method 2 The eluting solvent used in Method 1. was increased to 500 mL without fractionation. Table 2. indicates that recoveries of all six pesticides were obtained within the single elution in the range from 43.28 to 113.39%. Furthermore, organophosphorus pesticides showed more tendency of being recovered by this eluting method over the organochlorines, and malathion was the most recovered species (113.39%). Table 2. %Recovery of each studied pesticide obtained by a single eluting solvent on Florisil column Pesticides 2,4’-DDT 2,4’-DDE 2,4’-DDD Malation Methyl parathion Ethyl paration

3.3

%Recovery

43.28 56.41 52.72 113.39 62.35 80.95

Method 3

In this method, eluting solvent volume was set to 50 mL in each fraction, while acetone used inMethod 1. and 2. wasreplaced by ethyl acetate. Moreover, silica gel column was employed instead of Florisil. Results in Table 3. indicated that most organochlorine pesticides were almost completely recovered when the hexane containing 30% ethyl acetate was used. However, significant amount of 2,4’-DDE and 2,4’-DDT were still found in 100% ethyl acetate fraction. On the other hand, 100% ethyl acetate was the only fraction that eluted 85.14% of methyl parathion, while 40.74% malation and 82.24% ethyl parathion were recovered by 50% and 70% ethyl acetate in hexane, respectively. Table 3. %Recovery of each studied pesticide obtained by each fractionated eluting solvent on silica gel column %Recovery in each fraction Pesticides

2,4’-DDT 2,4’-DDE 2,4’-DDD

FRACTION1: FRACTION2: 50mL 30% 50mL 50% ethyl ethyl acetate in acetate in hexane hexane 87.78 ND 64.73 ND 107.61 ND 279

FRACTION3: 50mL 70% ethyl acetate in hexane ND ND ND

FRACTION4: 50mL ethyl acetate 4.95 17.60 ND

Total

92.73 82.33 107.61

Malation ND Methyl ND parathion Ethyl ND paration ND = Not detectable :8 WI

40.74 ND ND

ND

ND

85.14

ND

:4 :2 WI WI

40.74

82.24 ND

:4 WI

85.14 82.24

:8 WI

mVolt s

File: f:\star\data\no fraction.run Channel: A = A Results Last recalc: 8/13/2011 10:39 A M

500

400

300

200

F DDT

DDE

D E DDD

para-ethyl

malathion

para-methyl

A BC 100

0

-52

X: 0.8084 Minutes Y: 0.170 mV olts

5

10

15

20

25

30 Minut es

Figure 2. GC chromatogram of recovery test after elution by a single eluting solvent (Method 2.) A=methyl parathion, B=malathion, C=ethyl parathion, D=2,4’-DDE, E=2,4’-DDD, F=2,4’-DDT

In addition to the extraction procedure, chromatographic separation of all studied pesticides was tested within a single injection. It is obvious that organochlorine pesticides are a lot more sensitive to electron capture detection than organophosphorus pesticides. However, results from the GC-ECD analyses indicate that the single injection can provide a reasonable separation of all six pesticides (Fig. 2). 4.

Conclusion

Possibility of multi residue analyses of pesticides by a single method was evaluated by comparison of recoveries obtained from different elution on silica gel and Florisil column. Among the results obtained by three different eluting methods, silica gel was found to be the most preferable cleaning up column for all six studied pesticides according to the satisfied recoveries. In addition, ethyl acetate is also preferable slightly polar solvent to polarize hexane over acetone. The evaluation of the proposed study was successful in order to select the suitable conditions for further studies of multi residue analyses of common pesticides in food samples. 5.

Acknowledgement

The authors acknowledge the main support of the International Science Program, ISP, Uppsala University, Sweden for all funding throughout the works; National University of Laos for the facilities; and also Toyohashi University of Technology, Japan for the support of resource and consumable facilities. 6.

Reference The method used throughout this work was modified from Food and Agricultural Materials Inspection Center (FAMIC), Kobe, Japan. (http://www.famic.go.jp) 280