DEVELOPMENT OF A MULTI-RESIDUES METHOD FOR THE ANALYSIS OF PESTICIDES IN PEPER (PIPER NIGRUM)

Ching Lee Fung

Master of Science (Analytical Chemistry)

2015

DEVELOPMENT OF A MULTI-RESIDUES METHOD FOR THE

ANALYSIS OF PESTICIDES IN PEPPER (PIPER NIGRUM)

CHING LEE FUNG

A thesis is submitted

In fulfillment of the requirements for the degree of

Master of Science

(Analytical Chemistry)

Faculty of Resource Science and Technology

UNIVERSITY MALAYSIA SARAWAK

2015

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ACKNOWLEGDEMENTS

I wish to thank the Ministry of Science, Technology and Innovation (MOSTI) for financial

support to conduct this project; to Senior Research Officer Dr. Alvin Chai Lian Kuet of

Agriculture Sarawak (ARC) for permission to use the Pesticide Residue Laboratory facilities

and resources. My sincere thanks go to my supervisor, Professor Dr. Lau Seng and my co-

supervisor, Dr. Alvin Chai Lian Kuet, for their advice and guidance in carrying out this study

and for their invaluable comments, discussion and constructive criticism in reading the

manuscripts. Technical assistance rendered by the staffs of the Pesticide Residue Laboratory

is gratefully acknowledged. My sincere appreciation is also extended to all my friends who

have assisted in one way or another during this study.

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Development of a Multi-Residues Method for the Analysis of Pesticides in Pepper (Piper

nigrum)

Abstract

A rapid, specific and sensitive multi-residues method has been developed for the analysis of

6 pesticides in black pepper based on the Quick, Easy, Cheap, Effective, Rugged and Safe

(QuEChERS) solid-phase extraction (SPE) sample preparation method. In this work,

QuEChERS method, which was originally developed for vegetable samples with high amount

of water, was modified to improve the extraction of a wide range of pesticides in dried

product such as black pepper. The residues are extracted from the sample with acetonitrile.

The extracted organic phase containing the pesticides was further clean-up by eluting with

miniaturized carbon activated column (for organophosphorous pesticides) or silica column

(for organochlorine pesticides). The SPE method was chosen because less solvent is used,

time for clean up can be significantly reduced and cross contamination can be minimized.

The final extracts were analyzed by gas chromatography (GC) equipped with flame

photometric detector (FPD for organophosphorous pesticides) or electron capture detector

(ECD for organochlorine pesticides) or high performance liquid chromatography (HPLC)

equipped with ultra-violet detector (for benomyl). The recoveries of pepper samples spiked at

0.1, 0.5 and 1.0 mg kg-1 were between 60% to 75% with relative standard deviation (RSD) of

less than 8%. This extraction method provided a well-defined phase separation without

dilution and achieved acceptable recoveries. This method was applied for screening of

pesticide residues in peppers from Balai Ringin, Semongok and Malaysia Pepper Board.

Key words: solid-phase extraction, activated carbon powder cleanup, organochlorine,

organophosphorous.

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Pembangunan Kaedah Pelbagai-Sisa untuk Analisis Racun Serangga dalam Lada (Piper

nigrum)

Abstrak

Satu kaedah analisi pelbagai-residu yang cepat, spesifik dan sensitif telah dibangunkan untuk

analisis 6 racun serangga dalam lada hitam berdasarkan kaedah penyediaan sampel Quick,

Easy, Cheap, Effective, Rugged and Safe (QuEChERS) solid-phase extraction (SPE). Dalam

penyelidikan ini, kaedah QuEChERS yang asalnya digunakan untuk sampel sayur yang

mempunyai kandungan air yang tinggi, telah diubahsuai untuk meningkatkan pengekstrakkan

racun serangga dalam produk kering seperti lada. Sisabaki racun serangga diekstrak dengan

asetonitril . Fasa organik (ekstrak) yang mengandungi racun serangga kemudiannya

dibersihkan dengan turus mini karbon teraktif (untuk racun serangga organofosforus) dan

turus mini silika buatan-makmal (untuk racun serangga organoklorin). Kaedah SPE dipilih

kerana kurang penggunaan bahan pelarut, menjimatkan masa dan pencemaran silang dapat

dielakkan. Hasil akhir extrak dianalisa dengan gas kromatografi (GC) yang dilengkapi

dengan Flame Photometric Detector FPD (racun serangga organofosforus), Electron

Capture Detector ECD (racun serangga organoklorin) serta High Performance Liquid

Chromatography (HPLC) yang dilengkapi dengan ultra-violet detector (benomyl). Analisis

pemulihan sampel lada hitam pada pelbagai aras (0.10, 0.50 and 1.00 mg kg-1) adalah di

antara 60% hingga 75% dengan standard deviasi relatif (RSD) kurang daripada 8%.

Kesimpulannya, ia terbukti sebagai kaedah pengestrakkan yang universal kerana ia

memberikan fasa pembahagian yang jelas serta memberikan purata pemgembalian yang

boleh diterima. Kaedah yang dicadangkan telah diaplikasikan dalam permantauan racun

serangga pada lada hitam dari Balai Ringin, Semongok dan Kementerian Lada Malaysia.

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Kata kunci: pengesktrakan fasa pepejal (SPE), pembersihan dengan serbuk karbon teraktif,

organoklorin and organofosforus.

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TABLE OF CONTENTS

ACKNOWLEDGEMENT ii

ABSTRACT iii

ABSTRAK iv

LIST OF FIGURES x

LIST OF TABLES xii

LIST OF APPENDICES xiv

ABBREVIATION xv

CHAPTER 1: GENERAL INTRODUCTION

1.1 Pepper background and its applications 1

1.2 Pesticides in crops 4

1.3 Maximum Residue Limits (MRLs) 6

1.4 Problems and challenges in pesticide residues analysis in herbs and spices 8

1.5 Emergence of QuEChERS method 10

1.6 Objectives 11

CHAPTER 2: LITERATURE REVIEW

2.1 Pesticides information profiles 13

2.1.1 Methamidophos 14

2.1.2 Malathion 15

2.1.3 Chlorpyrifos 16

2.1.4 Quinalphos 18

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2.1.5 Cypermethrin 19

2.1.6 Benomyl 20

2.2 Residues Analysis 21

2.2.1 Matrix interference 22

2.2.2 Sample preparation and extraction techniques 23

2.2.3 Original QuEChERS method (Anastassiades et al., 2003) 26

2.2.4 Clean up techniques 29

2.2.4.1 Adsorption chromatography 29

2.2.4.2 Solid Phase Extraction (SPE) 30

2.2.4.3 Dispersive solid phase extraction (d-SPE) 33

2.3 Sorbent materials

2.3.1 Chemical and physical properties of silica 35

2.3.2 Activated carbon 36

2.4 Pesticide residues determination 37

CHAPTER 3: METHODOLOGY

3.1 Approach 40

3.2 Material 44

3.2.1 Reagents and Chemical 44

3.3 Sample preparation

3.3.1 Sample collection 44

3.3.2 Extraction 44

3.3.3 Clean up 45

3.3.3.1 Cleanup with loose sorbent (activated carbon powder)

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in sample vials – dispersive solid phase extraction 45

3.3.3.2 Cleanup with sorbent (activated carbon powder)

packed in pipette (column) – solid phase extraction 45

3.3.3.3 Clean up by silica gel with 10% water content

(Miyahana et al., 1993) 46

3.4 Instrumental analysis

3.4.1 Determination of pesticide residues retention time (tR) 50

3.4.2 OP pesticides analysis 50

3.4.3 OC and PY pesticides analysis 52

3.4.4 Benomyl analysis 53

3.5 Method validation

3.5.1 Spike and recovery 54

3.6 Application of method on field samples 56

CHAPTER 4: RESULTS AND DISCUSSION

4.1 Determination of pesticide residues retention times (tR) 57

4.2 Sample preparation and extraction 59

4.2.1 Dry extraction (acetonitrile) 59

4.2.2 Wet extraction (mixture of acetonitrile and water) 61

4.2.3 Repeated extraction 63

4.3 Cleanup

4.3.1 Absorption of pesticides to activated carbon 64

4.3.2 Recovery study using clean-up by dispersive-SPE 66

4.3.3 Recovery study using clean up by miniaturized activated

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carbon SPE column 69

4.3.4 Recovery study on OC and PY pesticide using silica gel 71

4.4 Applications 74

CHAPTER 5: CONCLUSIONS 76

5.1 Activated carbon d-SPE 78

5.2 Activated carbon miniaturized SPE column 79

5.3 Silica gel cleanup 79

REFERENCES 82

LIST OF APPENDICES 92

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LIST OF FIGURES

Figure 1 – (a) Raw pepper berries (b) Processed black and white peppers 3

Figure 2 – Chemical structure of methamidophos 14

Figure 3 – Chemical structure of malathion 15

Figure 4 – Chemical structure of chlorpyrifos 17

Figure 5 – Chemical structure of quinalphos 18

Figure 6 – Chemical structure of cypermethrin 19

Figure 7 – Chemical structure of benomyl 20

Figure 8 – Traditional approach for multi residue analysis in foods. 25

Figure 9 – QuEChERS pesticide analysis workflow (Anastassiades et al., 2003) 26

Figure 10 – Method development from QuEChERS pesticide analysis work flow used in this

research. 34

Figure 11 – Schematic cleanup procedure with loose sorbent (activated carbon powder)

in sample vials (mechanical shaking) – dispersive solid phase extraction. 47

Figure 12 – Schematic cleanup procedure with sorbent (activated carbon powder) packed

in pipette (column) – solid phase extraction. 48

Figure 13 – Schematic cleanup procedure with sorbent (silica gel) packed in pipette (column)

– solid phase extraction 49

Figure 14 – Calibration curve for methamidophos in pepper 55

Figure 15 – Calibration curve for malathion in pepper 56

Figure 16 – GC chromatogram of OP pesticides : methamidophos (3.281 min);

malathion (10.759 min); chlorpyrifos (11.154 min); quinalphos (12.811 min). 58

Figure 17 – GC chromatogram of OC pesticide standard : cypermethrine 1,2,3

(22.228 min, 22.430 min, 22.650 min). 58

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Figure 18 – HPLC chromatogram of benomyl (detected as carbendazim) with retention

time, tR, of 4.867. 59

Figure 19 – GC chromatogram of OP pesticides (0.1 mg kg-1) after cleanup using

dispersive SPE activated carbon. 68

Figure 20 – GC/ECD chromatograms of pepper samples spiked with cypermethrin at

1.0 kg mg-1. A: activated carbon cleanup; B: silica gel cleanup 73

Figure 21 – Summary of research method development for pesticides analysis in Piper

nigrum. 81

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LIST OF TABLES

Table 1 – Summary which highlight the modifications made to the original QuEChERS and

the justifications for the respective modifications. 43

Table 2 – The condition of OP analysis instrument Hewlett Packard 5890 Series

with Flame Photometric detector II GC (FPD). 51

Table 3 – The condition of OC analysis instrument Hewlett Packard 5890 Series II

GC with Electron Capture detector (ECD). 52

Table 4 – The condition of HPLC, Agilent 1100 HPLC with ultra-violet DAD

detector used to analysed benomyl. 53

Table 5 – Calibration curves results 55

Table 6 – The retention times, tR, of the pesticides used in this study. 57

Table 7 – Recovery values for pesticides extracted with acetonitrile solvent

(dry extraction). 60

Table 8 – Recovery data for pesticides extracted with acetonitrile/water mixture

(60/40, v/v) (single extraction). 62

Table 9 – Recovery data for pesticides extracted twice with acetonitrile/water mixture

(60/40, v/v) (Repeated extraction). 64

Table 10 – Mean recovery value (%) obtained for different amount of activated

carbon (mg) used in dispersive SPE. 65

Table 11 – Mean recovery value (%) obtained for different amount of activated carbon

(mg) used in column SPE. 66

Table 12 – Mean recovery values (%) obtained using dispersive-SPE (activated carbon)

cleanup method. 67

Table 13– Recovery data for pesticides determined by activated carbon SPE column. 70

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Table 14– Recovery data for cypermethrin determined by silica gel clean up (n=9). 72

Table 15– Recovery results from field samples analyzed with proposed and traditional

multi-residues method. 75

Table 16– Advantages of the proposed method compared with the existing system. 77

Table 17– Comparison between traditional and proposed multi-residues methods. 77

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LIST OF APPENDICES

Appendix 1: Recovery values for pesticides extracted with acetonitrile solvent (dry

extraction). 92

Appendix 2: Recovery data for pesticides extracted with acetonitrile/water mixture

(60/40, v/v) (Single extraction). 94

Appendix 3: Recovery data for pesticides extracted twice with acetonitrile/water

mixture )60/40,v/v) (Repeated extraction). 96

Appendix 4: Mean recovery value (%) obtained for different amount of activated

carbon (mg) used in dispersive SPE. 98

Appendix 5: Mean recovery value (%) obtained for different amount of activated

carbon (mg) used in column SPE. 100

Appendix 6: Mean recovery value (%) obtained using dispersive-SPE (activated

carbon) cleanup method. 102

Appendix 7: Recovery data for pesticides determined by activated carbon SPE column. 105

Appendix 8: Recovery data for cypermethrin determined by silica gel clean up (n=9). 108

Appendix 9: Statistical analysis (ANOVA) – Method evaluation. 109

Appendix 10: Statistical analysis (ANOVA) – Pesticides recovery evaluation. 111

Appendix 11: Calibration curves 114

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ABBREVIATIONS

Å angstrom

ANOVA analysis of variance

ARC agriculture research center

ASE accelerated solid extraction

d-SPE dispersive solid phase extraction

ECD electron capture detector

FPD flame photometric detector

GC gas chromatography

GPC gel permeation chromatography

HPLC high performance liquid chromatography

KCI potassium chloride

LC liquid chromatography

LOD limit of detection

MgSO4 magnesium sulphate

MPB Malaysia Pepper Board

MRL maximum residue limit

MSPD matrix solid phase dispersion

NaCI sodium chloride

OC organochlorine

OP organophosphorous

PY pyrethroid

PSA primary secondary amine

QuEChERS Quick, Easy, Cheap, Effective, Rugged and Safe

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SBSE stir-bar sorption extraction

SD standard deviation

SFE supercritical fluid extraction

SiO2 silica

SPE solid phase extraction

SPME solid phase microextraction

tR retention time

UV ultra violet

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CHAPTER 1

GENERAL INTRODUCTION

1.1 Pepper background and its applications

Human diet constitute of 5 main nutrition primarily protein, carbohydrate, fiber, fat

and minerals. Nevertheless, to further enhance the flavor and aroma of food, herbs and spices

are added in small quantity as they are also part of essential constituents of human diet.

Early documentation suggests that hunters and gatherers wrapped meat in the leaves of

bushes, accidentally discovering that this process enhanced the taste of the meat. They later

discovered as well that certain nuts, seeds, berries, and bark also improved the taste of food.

Spices and herbs were also used as a way to mask unpleasant tastes and odors of food, and

later, as food preservation process to keep food fresh (Tapsel et al., 2006).

Unlike earlier times when monopolies dominated the spice trade, commerce in spices

is now relatively decentralized. Throughout the world, spices and herbs are frequently used in

cuisine, largely to improve flavor and to provide new tastes. Today, people are increasingly

interested in enjoying spices and herbs for health benefits.

In order to fulfill consumers’ demand, the use of pesticides (insecticides and

herbicides) plays an important and essential role in increasing the world production of herbs

and spices. However, the pesticides should be used cautiously to ensure that they do not

remain in the crops, which may later create potential harm to human health as well as the

environment.

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As Malaysia is a multi-racial country, herbs and spices are easily accessible and

available in the market. Malaysia has a tropical climate, with very warm days and fairly cool

breeze nights. On average, the temperature holds at 30°C (86°F), with cooler temperatures in

the highlands. With this climate, it is suitable to cultivate some of the herbs and spices such as

pepper, chili peppers, garlic, ginger, curry leaves, basil, turmeric and so on.

Since its discovery by European consumers in the seventeenth century, pepper is the

main spice produced and traded worldwide for both its flavor and its use as a medicine. The

two major types are black and white pepper, and both are generally traded as whole spice.

World production and exports of pepper are quite concentrated where India, Indonesia,

Malaysia, Thailand and Brazil account for 87% of world production and 82% of world

exports (UNCTAD, 1995).

Pepper is one of the oldest and most important spices in Malaysia. It is derived from

berries of tropical plant named Piper nigrum. It is a type of spice that has been exploited

widely as food enhancer in Malaysia. In Sarawak alone, pepper had been cultivated since year

1856 but vast cultivation only started in the 1900s. At the present, pepper is still the main

plant grown as a source of income for rural residents in Sarawak.

Pepper is being planted in districts around Sarawak; these were Kuching, Samarahan,

Sri Aman, Betong and Sarikei. A rough estimation of land needed for pepper cultivation in

Sarawak is around 13,000 hectares. With man power and lands available for cultivation, more

that 98 percent of total pepper production in Malaysia came from the state of Sarawak. In

2004, Malaysia produces 20,000 metric tons of pepper in which 19,748 metric tons that worth

RM 113.2 million have been exported to other countries (UNCTAD, 1995).

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Black pepper is produced by drying the matured berries under the sun while white

pepper is processed by soaking the berries in clean running water to remove the husk before

drying under the sun.

Figure 1: (a) Raw pepper berries (b) Processed black and white peppers

Figure 1 shows raw pepper berries and its end products after being processed. The

small, dark, unripe fruit of the pepper plant (Piper nigrum), is being used whole or ground as

a pungent spice and seasoning. Pepper gets its spiciness mostly from the piperine chemical

compound, which is found both in the outer fruit and in the seed.

In Sarawak where black peppers are mainly grown, pepper trees are being protected

against various pests and diseases largely by chemical means. Those more extensively used

belong to the class of organophosphorous (OP), organochlorine (OC) and pyrethroid (PY)

insecticides. Methamidophos, quinalphos, cypermethrin, chlorpyrifos, malathion, and

benomyl are some of the most frequently used pesticides in pepper trees treatment.

During cultivation, pepper berries receive direct applications of pesticides in the field

and small portion of the pesticide residues is likely to remain at harvest. Therefore, regular

(a) (b)

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monitoring programme has to be carried out to prevent consumer from over exposure to

harmful pesticide residues in the diet.

Pesticides are used by farmers to control weeds and prevent crop damage by insects,

rodents as well as fungus attack. Pests and diseases can cause damages to pepper vines and

thus hinder its growth. The main pests that damage the vine and inhibit the development of

flower spikes are pepper weevil (Lophobaris piperis) and tingid bugs (Dicronocoris hewetii).

Besides pests, fungus such as Phytophthora capsici, Colletotrichum capsici, Colletotrichum

piperis and Colletotrichum gloeosporioides which cause diseases, grown in hot and humid

country can restrain pepper vines from growing healthily (Paulus et. al, 2007). During

storage, peppercorns can also be attacked by cigarette beetle (Lasioderma serricorne).

Therefore, pesticides were also being used on pepper after harvest to prolong their storage

life.

1.2 Pesticides in crops

Generally, pesticides helps to control weed species, harmful insects and numerous

plant diseases that upset the crops. Without these important crop protection and pest control

technologies, food production would decline, resulting fruits and vegetables in short supply.

Eventually, as the condition persist, the price of food commodities would rise tremendously.

For many years, we have enjoyed the benefits of using pesticides to control weed,

insect, fungus, parasitic, and rodent pests. But the usage of pesticides can also cause potential

risks to the environment as well as human health. Potentially harmful impacts of pesticides

include acute poisoning from a short-term exposure can result in death. Chronic impacts of

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long-term exposure to pesticides, including pesticide residues in food, could also result in

death.

Organochlorine (OC), organophosphorous (OP) and pyrethroid pesticides are the three

main pesticides groups which have a long history of widespread and extensive usage around

the world. OC pesticides are insecticides composed primarily of carbon, hydrogen, and

chlorine. These compounds are typically very persistent in the environment and are known to

accumulate in sediments, plants and animals. Most of them break down slowly and can

remain in the environment long after application and in organisms long after exposure

(EUROPA, 2001). As OC compounds break down slowly, they tend to build up in fatty

tissues and remain in our bodies for a long time.

Because of OCs’ high persistency in the environment, the agrochemical industry have

turned to the less persistent, but more acutely toxic organophosphorous (OP) and pyrethroid

(PY) compounds to control insect pests on crops. The usage of these pesticides has increased

rapidly, and today about 79% of insecticides applied nationwide are OPs and PY compounds.

The OP pesticides work by interfering with the nervous system of insects, a mechanism that

also affects the human nervous system when people are exposed.

Some pesticides are no longer permitted for use because of their toxic effects or cancer

causing potential. Unfortunately, some of these descriptions are unavoidable in food. This is

because persistent pesticides still can be found in the environment many years after their use

have been stopped. These residues are sometimes found in food crops grown on contaminated

soil. These pesticides may remain in small amounts (called residues) in or on fruits,

vegetables, grains, and other foods.

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Under Food Act 1983, tolerances are set to enforce lowest possible levels for such

unavoidable pesticide residues. If a food is found to have levels of the pesticides higher than

these tolerance levels, federal or local officials can seize and remove the food before destroy

it. This monitoring programme can ensure the consumers do not consume tainted food which

can deteriorate health.

However, there are some pesticide residues detected in food for which no tolerances

have been set. This could due to the pesticides being recognized by the Environment

Protection Agency (EPA) as safe and not requiring any tolerance. It could also due to the

pesticides not being registered for agricultural use. But more often, the reason for pesticides

with no tolerances set is due to the pesticides is on a crop different from the one for which it

has been approved.

Thus, more tolerances or maximum residue limits (MRLs) for pesticides should be

established on variety of food commodities as a guideline to ensure the safety of food

consumption is guaranteed.

1.3 Maximum Residue Limits (MRLs)

Public concern in recent years about health risks arising from pesticide residues in the

diet is alarmingly increasing. They require pesticide monitoring in agricultural produce to be

strictly carried out. Environmental Protection Agency (EPA) is an international agency that

responsible in setting tolerances or maximum residue limits (MRLs) for pesticides. A

tolerance is the maximum amount of a specific pesticide or its break down products

(metabolites) that is allowed to remain in or on foods. In Sarawak, the monitoring of pesticide

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residues in crops is carried out by the Department of Agriculture and Ministry of Health. The

key purpose of the residue monitoring is to ensure that our food contains only the permitted

level or maximum residue limits (MRLs) of pesticide residues.

Pesticide residue levels in the treated crops are regulated through the establishment of

Maximum Residue Levels (MRLs). An MRL is the maximum concentration of a pesticide

residue, (mg kg-1), legally permitted in or on food commodities. When pesticide residues in

food exceeded the MRLs, the produce concerned will be removed from the market and

destroyed. The frequent decrease in the maximum residue limits (MRLs) imposed by

international organizations (European Union, Environmental Protection Agency) implies a

dramatic improvement of the limits of detection provided by currently available multi-

residues methods.

Regular monitoring programme has to be carried out by regulatory agencies to prevent

the consumer from over exposure to harmful pesticide residues in their diet. This is also a way

to exert pressure on growers to produce safer crops, avoid the use of banned pesticides, over

dosage and abuse in pesticide application.

In an effort to monitor the levels of these residues, many governmental and industrial

programs have been implemented for the regulatory analysis of pesticide residues in food

through multi-residues methods to detect many compounds of specific chemical classes.

These analytical procedures were developed by numerous governments and private

laboratories throughout the world (Okihashi et al., 2005). A large number of pesticides can be

determined by the various multi-residues screens.