Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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continuous belt, rotary wiper, and Rogueing glove

application. The Rope-wick device is a rig consisting

of a series of short, exposed nylon ropes, each end of

which is connected to a reservoir of herbicide solu-

tion. The solution passes into the ropes by both capil-

lary action and gravitational flow. As the applicator

moves through the weed-infested field, the chemical

on the soaked wicks is rubbed on to the tops of the tall

weeds but not on to lower-growing crop. Spray drift

is eliminated. The carpet roller, as the name suggests,

is a tractor mounted with a nylon carpeted roller

soaked with liquid herbicide through a delivery

system from a herbicide reservoir. The design is such

that herbicide will only be in contact with the tall

weeds without contacting the desired crop. A con-

tinuous belt consists of a V-belt, with sponges glued

on to it, passing through a herbicide-containing reser-

voir. An adjustable pressure wheel removes excess

herbicide from the sponges prior to its application

on to the weeds. This helps prevent herbicide from

contacting the crop. A rotary wiper applicator con-

sists of flexible arms that allows wiper rotation

around stationary objects to avoid injury to tree

trunks. A glove equipped with absorbent pads that

has continuous loading of herbicide solution from a

reservoir is known as the Rogueing glove application.

0011 A relatively new way of applying herbicide is

through a sprinkler system. Liquid herbicide is pumped

into the system and sprayed in a manner similar to a

water sprinkler system.

0012 Aerial spraying, using small aircraft or helicopters,

can cover a large field in a shorter time period.

Sprayers similar to those used in the ground operation

can be mounted on to the aircraft for application to

large areas.

0013 Considerable research has been conducted by chem-

ical companies to devise controlled-release formula-

tions, a technology which has been demonstrated to be

safe and effective in the drug industry. The three con-

trolled-release systems are: (1) the chemically bound

system, in which the herbicide is bound to a polymer;

(2) the microencapsulation system, where the herbi-

cide is coated by a polymer; (3) the matrix encapsu-

lation system, where the herbicide is dispersed within

a polymer matrix. This technology renders safer herbi-

cide handling, reduces environmental pollution, and

enhances herbicide selectivity.

0014 The treatment of seeds with herbicides is another

area of research currently under investigation. Cer-

tain herbicides can be applied to certain crop seeds

prior to planting. Once planted in the soil, the her-

bicide moves swiftly away from the seed to the

surrounding area to inhibit weed growth. This ap-

proach is economical and convenient to use. It also

has a lesser tendency to pollute the environment.

Safety Implications

0015Ideally, herbicides are chemicals designed to cause

injury only to undesirable weeds. Due to the biological

differences between plants and animals, herbicides

have low acute toxicity towards humans. However,

most chemicals tend to have more than one metabolic

effect on different living organisms. It is such unex-

pected secondary or side-effects of chemicals that

cause major concern. Before the approval for

marketing of any herbicide by the government, chem-

ical companies must conduct extensive studies on the

effectiveness of new herbicides. In addition, studies on

metabolic fate and toxicity to animal species and

plants are conducted to insure that the herbicide is

safe to be used in the field. The cost of these testing

procedures is tremendous. The specific requirements

for herbicide registration depend upon the law of the

country in which they are used but, in general, herbi-

cides may not be used without registration by some

governmental agency that carries out extensive

reviews of the studies and tests performed by the

chemical companies. It is also the duty of the vendor

to provide users with information about all precau-

tions and safe handling procedures of the herbicide.

Adherence to those instructions by users is of utmost

importance for the avoidance of harmful exposure.

0016The health effects of herbicides and their environ-

mental impacts have been a major concern of the

public. The health and safety issues associated with

herbicide use affect not only consumers, but also

farmers, formulators, applicators, and field workers,

as well as the users of home and garden products.

Groundwater contamination by agricultural chem-

icals has been an issue worldwide and will continue

to be important in the years to come. Restrictions on

the use and banning of certain herbicides are on the

rise. Development of biodegradable herbicides with

low residue levels combined with improved delivery

systems can help protect the environment.

0017Depending on the conditions, exposure to herbi-

cides at a high concentration can be fatal. Precautions

in handling herbicides should be strictly followed to

avoid unnecessary exposure. Safety training of all

personnel designated to handle herbicides is impera-

tive, and refresher safety training should also be con-

ducted periodically to keep personnel up-to-date on

all safety issues.

0018Equipment for application should be inspected fre-

quently to insure that it is functioning properly. Mal-

functioning equipment should be repaired prior to

usage. Emergency procedures covering spillage or ac-

cidental poisoning should be established and strictly

followed. There should be health surveillance pro-

grams for workers to monitor and insure worker

PESTICIDES AND HERBICIDES/Types, Uses, and Determination of Herbicides 4485

well-being. Personal protective equipment and

clothing should be checked for leaks periodically

and should be kept clean. Good record-keeping of

herbicide inventory and application is necessary to

account for usage.

0019 In order to avoid inhalation of herbicides during

application, workers should wear respirators. There

are many different kinds of respirators, namely the

chemical cartridge respirators, powered air-purifying

respirators, canister respirators, supplied-air respir-

ators, and self-contained breathing apparatus. There

are also certain herbicides that can be absorbed by the

skin; users should handle these with due caution and

should wear appropriate protective clothing, includ-

ing face shields.

0020 Care should be given in the storage and transport of

herbicides to minimize spillage. In case spillage occurs,

proper decontamination should be performed imme-

diately. Maintenance of all equipment for dispensing

herbicides should be done routinely. Warnings should

be given in advance to alert others of possible herbi-

cide drift during application, and warning signs and

restricted-entry signs should be posted to prevent

others from entering the treated areas.

Specific Examples of Uses

0021 Weeds are usually defined as undesirable plants. Weeds

are often the primary concern of farmers, because

they cover many millions of productive acres that

could be used to grow beneficial crops. In the past,

farmers controlled weeds by manually removing them

from the crops. The ancient Romans killed weeds

with salt. With farms of small size, manual weed

control, such as hand hoeing and pulling, mowing,

burning, and machine tillage, is feasible. However,

with large farms such labor is extensive and costly.

As the size of the farms increased and synthetic herbi-

cides were introduced, farmers began to rely on herbi-

cides to control weeds.

0022 Generally, there are three different types of treat-

ment for the application of herbicides. They are

preplanting, preemergence, and postemergence treat-

ments. Preplanting treatment takes place prior to

planting; preemergence treatment is done after

planting but preceding the crop or weed emergence;

and postemergence treatment is performed after the

emergence of the crop or weeds.

0023 2-4-Dichlorophenoxyacetic acid (2,4-D) was one

of the first synthetic herbicides introduced to control

broad-leafed weeds in cereal crops and pastures. 2,4-

D is an effective systemic herbicide and is selective for

broad-leafed plants but not grasses. It can be used as

either a pre- or a postemergence herbicide for corn,

but only as postemergence for sorghum. 2,4-D is

highly versatile and is used on a variety of crops

such as wheat, barley, oats, rice, and sorghum.

During the Vietnam war, Agent Orange, a 50: 50

mixture of 2,4-D and 2-4-5-Trichlorophenoxyacetic

acid (2,4,5-T), was used extensively over the terrain

in Vietnam as a defoliant to clear the way for US

troops. 2,4,5-T is usually contaminated with dioxin,

a highly toxic chemical compound and known car-

cinogen. It is due to this notorious contaminant that

Agent Orange has been blamed for various illnesses

and reproductive problems among those who came in

contact with the defoliant in Vietnam.

0024Paraquat is used as a preemergence treatment for

sugar beet. Simazine, on the other hand, is used both

as a preplant and as a preemergence treatment

for corn. S-ethyl dipropylthiocarbamate (EPTC) is

incorporated into the soil as a preplant treatment

for potatoes.

0025Herbicides have made it possible to grow more

food on less land with less labor and at lower cost.

Herbicides are also used to control aquatic weeds

which impede water flow in irrigation canals and

drainage systems, interfere with fishing, or promote

insect-breeding grounds.

Stability in the Environment

0026For herbicides to exert their effects on weeds, they

must be relatively stable in the treated environment.

However, the stability of the chemical creates a burden

on the environment, especially for those herbicides

that find their way into aquifers and contaminate

drinking water sources or remain on the crops at the

time they reach consumers. In order to insure that the

newer generation of herbicides do not linger on after

accomplishing their task, research is directed towards

synthesizing biodegradable compounds. The ideal

herbicide is one that degrades to harmless chemicals

after it performs its function and therefore does not

persist in nature. Carbamates are one such class of

chemicals specifically designed with that goal in mind.

Analysis of Residues in Foods

0027There is increasing awareness among consumers of

the hazard of chemical contamination of food and

drinking water. There is particular concern over the

implications of food contamination by herbicide resi-

dues. Analysis for herbicide residues in food requires

methods that identify not only parent structures, but

also their metabolites and degradation products in a

variety of food matrices. Certain food crops are per-

ishable and therefore cannot wait for lengthy analysis

to establish the suitability for consumption. Thus,

rapid analytical technology is needed. Multiresidue

4486 PESTICIDES AND HERBICIDES/Types, Uses, and Determination of Herbicides

methods, which can detect the presence of many herbi-

cides at once, are the methods of choice for determin-

ing the presence of a multiple number of herbicides

and their degradation products in a food sample.

0028 An analytical process consists of several major steps:

the sample preparation, the extraction, the clean-up,

the determinations, and the confirmation. These steps

are common to the determination of other agrochem-

ical residues, including pesticides, and are discussed in

detail in the following article. The basic operation of

sample preparation is to separate physically food or

plant parts and to chop and blend them. The essence of

the extraction process is to remove the target herbicide

from the other components in the sample matrix. The

main function of the clean-up procedure is to remove

interfering constituents, usually by selective partition-

ing into organic solvents followed by an adsorption or

size exclusion chromatographic purification step. The

determination step includes separation of the purified

samples through thin-layer chromatography, gas chro-

matography, or liquid chromatography techniques

followed by the detection procedure using a variety of

specific detectors for the targeted compound. For con-

firmation purposes, the analyte is further subjected

to mass spectrometric analysis. Recently, successful

attempts have been made in using gas chroma-

tography–mass spectroscopy (GCMS) as a primary

screening method. The GCMS screening technique

provides simultaneous results for both the detection

and the confirmation of the targeted compound in the

sample matrix. This one-step procedure will be the

method of choice as it offers both rapid and definitive

data. (See Chromatography: Thin-layer Chromatog-

raphy; Gas Chromatography; Mass Spectrometry:

Principles and Instrumentation.)

0029 Improvements to existing analytical technology

are well underway to reduce the time- and solvent-

consuming extraction and clean-up steps. Supercrit-

ical fluid extraction, which is based on the solvent

properties of gases such as carbon dioxide at its critical

pressure and temperature, can selectively remove the

targeted compound from the complex food matrix in a

short time. Through such approaches the recovery of

the compound can be easily accomplished.

0030 The use of antibodies as analytical tools is a

common practice in clinical laboratories. Antibodies

have been recently developed for identifying and

quantifying herbicides. Antibodies can be isolated

from the plasma of an immune-challenged animal or

from a hybridoma cell line. An antibody that is spe-

cifically generated from a compound will have high

selectivity towards that compound even in the midst

of other interfering components and can bind to it

tightly to form a complex. Therefore, by attaching a

tracer to the antibody molecule, one can quantify the

amount of antibody complex present, which is also an

indication of the amount of the compound in the

sample. A variety of tracers are available, for example

radioisotopes, fluorescent molecules, and so forth.

One of the disadvantages of the immunoassay is

that the length of time to generate the specific anti-

body is relatively long. Typically, it takes approxi-

mately a year to develop. However, once it is

generated, the immunoassay can be performed in

less than half an hour. The triazine immunoassay

is now available commercially, and, like most im-

munoassays, it is specific, sensitive, rapid, and cost-

effective. (See Immunoassays: Principles.)

0031One of the modes of action of herbicides is by

inhibiting photosynthesis. The Hill reaction is one of

the processes in the photosynthetic pathway; there-

fore, a screening technique based on the inhibition of

the Hill reaction can be a useful tool in detecting

herbicides like triazines and carbamates.

See also: Chromatography: Thin-layer Chromatography;

Gas Chromatography; Immunoassays: Principles; Mass

Spectrometry: Principles and Instrumentation;

Pesticides and Herbicides: Residue Determination

Further Reading

Ashton FM and Crafts AS (1981) Mode of Action of

Herbicides, 2nd edn. New York: Wiley.

FDA (1968) Pesticide Analytical Manual, 2nd edn, vol. 1.

Methods Which Detect Multiple Residues. Washington,

DC: US Department of Health Services, Food and Drug

Administration.

Klingman GC, Ashton FM and Noordhoff LJ (1982) Weed

Science: Principles and Practices, 2nd edn. New York:

Wiley.

McWhorter CG and Gebhardt MR (eds) (1987) Methods of

Applying Herbicides. Monograph Series of The Weed

Science Society of America, no. 4. Champaign, IL: The

Weed Science Society of America.

Newton M and Knight FB (1981) Handbook of Weed and

Insect Control Chemicals for Forest Resource Managers.

Beaverton: Timber Press.

Residue Determination

S Nawaz, Central Science Laboratory, Sand Hutton,

York, UK

Background

0001Continued population growth has led to an increased

demand on the world’s natural resources. Pesticides

are widely used to help increase the yield and improve

PESTICIDES AND HERBICIDES/Residue Determination 4487

the quality of crops. Pesticides are categorized

according to their mode of action and include insecti-

cides, herbicides, fungicides, acaricides, nematicides,

and rodenticides. Pesticides are also used as plant

growth regulators and for public-health purposes.

Global sales of pesticides during 1996 were estimated

at over US$30 billion. Herbicides, insecticides, and

fungicides accounted for 48, 28, and 18%, respect-

ively, of the total sales (see Table 1). There are over

900 chemicals registered for plant protection pur-

poses in the European Union (EU) alone. In addition

to the existing pesticides, there are an ever increasing

number of new chemicals being granted approval.

0002 As pesticide use can leave undesirable residues,

various national and international authorities regu-

late the use of pesticides and set maximum residue

levels (MRLs) in crops. An MRL is the maximum

concentration of a pesticide and/or its toxic metabol-

ites legally permitted in food commodities and animal

feeds. If pesticides are properly applied at the recom-

mended rates, and crops are only harvested after

the appropriate time intervals have elapsed, residue

levels are not expected to exceed MRLs. The residue

levels in foods derived from commodities that comply

with the respective MRLs are also intended to be

toxicologically acceptable. In the EU, the regulation

of the agrochemical industry, and the setting of MRLs

is currently being harmonized across all member

states by the EC. In the USA, the Environmental

Protection Agency is responsible for such regulation.

The Codex Alimentarius Commission (FAO/WHO)

has published tables of MRLs which have official

status across the world and are used to aid inter-

national trade. The monitoring of the residues in

foods is often at the microgram per kilogram level

or lower and has to be supported by strict analytical

quality-control standards, so that the analysis pro-

duces unequivocal, precise, and accurate residue

data. Before samples are analyzed, the analyst has to

demonstrate that the intended analytical method can

achieve adequate specificity, sensitivity, linearity, ac-

curacy, and precision at the relevant analyte concen-

tration and in appropriate matrices. The calibration

solutions must be prepared using certified reference

standards. Residue analyses normally include the

metabolites, isomers, and other related compounds

included in the MRL definition. Many methods can

determine a large number of residues in a single ana-

lytical run; these multiresidue methods are in common

use and help reduce the total cost of analysis.

Sampling

0003A representative sample consists of a large number of

randomly collected units. It is not always possible to

collect large samples because of the cost of transpor-

tation and the practicalities of sample handling in the

laboratory. Monitoring of pesticide residues for MRL

compliance involves analysis of a composite sample,

made up of a number of individual units. Recent

research has shown that pesticide residues in individ-

ual units of fruit and vegetables can exhibit an ex-

tremely skewed distribution, and this is likely to add

to the difficulty of taking a representative sample. The

guidelines for obtaining composite samples for MRL

compliance monitoring are published by the Codex

Alimentarius Commission and are summarized in

Table 2. In addition to checking for MRL compliance,

residue analyses are also carried out to investigate

other issues such as cases of misuse or the deliberate

poisoning of wildlife or domestic animals. In such

instances, a more targeted sampling regime may be

adopted, and a qualitative analysis may suffice.

Sample Preparation/Subsampling

0004Samples should be analyzed without any delay, as

some pesticide residues may degrade rapidly. If

tbl0001 Table 1 Major classes of chemicals used as pesticides

Type of chemical Examples and their primary uses

Benzimidazole Benomyl (F), carbendazim (F),

thiabendazole (F)

Bipyridylium Diquat (H), paraquat (H),

Carbamate Aldicarb (I, N), carbaryl (I),

carbofuran (A, I, N)

Dithiocarbamates Mancozeb (F), ziram (H), thiram (H)

Organochlorine DDT (I), lindane (I), endosulfan (I)

Organophosphorus Chlorpyrifos, malathion (I, A),

parathion (I)

Pyrethroids Permethrin (I), cyfluthrin (I)

Substituted phenyl ureas Diuron, linuron, monolinuron (H)

Triazine Atrazine (H), simazine (H)

A, acaricide; F, fungicide; H, herbicide; I, insecticide; N, nematicide.

tbl0002Table 2 Codex guidelines for collection of representative

samples

Sample type Minimumweight of sample (kg)

Small or light products

(e.g., berries, peas, spinach)

Medium sized products

(e.g., apples, carrots, potatoes)

1 (at least 10 units)

Large size products

(e.g., melons)

2 (at least 10 units)

Dairy products

(e.g., cheese, butter)

0.5

Meat, poultry, fish 1

Oils and fats 0.5

Cereals and cereal products 1

4488 PESTICIDES AND HERBICIDES/Residue Determination

immediate analysis is not possible, storage of samples

at 20



C may help minimize the degradation pro-

cess. Typically, a 20–50-g portion (subsample) is re-

quired for analysis. In order to obtain representative

subsamples, it may be necessary to grind/mill and

thoroughly mix the whole sample, so that any resi-

dues present are evenly distributed. This process is

especially important because residue levels can ex-

hibit a high degree of variability between individual

units. Some pesticides are known to degrade during

the processing of fruit and vegetable samples at ambi-

ent temperature. Milling frozen food samples in the

presence of excess solid CO

(dry ice) has been shown

to minimize the losses of most pesticide residues

during the process.

0005 Domestic mills (e.g., coffee grinder) as well as more

specialized mills are used to grind samples of cereals,

nuts, and pulses. Manual methods, such as cone and

quarter or mechanized devices such as the riffle div-

ider can be used to obtain representative subsamples

from such samples. Samples of animal tissues are

minced and mixed thoroughly before subsampling.

For pesticides (e.g., organochlorines) that accumulate

in the fatty tissue of animals, visible layers of fat may

be removed for direct analysis. Preparation of homo-

genous fruit and vegetable samples prior to sub-

sampling may be carried out using domestic food

processors and blenders. However, larger specialized

mechanical bowl choppers are more suitable for large

samples, and heavy-duty choppers may be required to

process frozen samples.

Extraction

0006 The extraction step involves the quantitative transfer

of pesticide residues from the food matrix into solv-

ent(s). The efficiency of extraction process depends

on the physicochemical properties of the solvent(s)

and analytes. Important factors include pH, polarity,

temperature, sample/solvent ratio, presence of water,

and degree of analyte/matrix binding. Most extrac-

tion procedures employ organic solvents in the pres-

ence of water. The presence of water is critical for

extraction of pesticides from cereals and cereal prod-

ucts, as it helps reduce the binding between residues

and matrix. Samples of cereals, nuts, pulses, fruit, and

vegetable samples are extracted by simple homogen-

ization with an organic solvent. The most commonly

used solvents include ethyl acetate, acetone, acetoni-

trile, hexane, methanol, and dichloromethane. For

multiresidue extraction methods, it is not possible to

establish the optimum extraction conditions for all

residues with differing physical and chemical proper-

ties so the choice of solvent polarity is usually a

compromise. Although the presence of water may

aid the extraction process, most polar pesticides are

partitioned into the water phase if the organic solvent

used is not water-miscible. Addition of anhydrous

sodium sulfate prior to extraction can overcome

this problem. The analysis of nonpolar pesticides in

fatty products involves extraction of fat using non-

polar solvents such as hexane, n-pentane, or light

petroleum. After the evaporation of the solvent, the

fat is redissolved in an organic solvent prior to the

clean-up and determination steps.

0007Other methods used for residue extraction include

soxhlet, where samples are exposed to solvent vapor

that is condensed and vaporized repeatedly to

exhaustively extract analyte(s). Supercritical fluid ex-

traction (SFE) methods use a gas under high pressure

and above the critical temperature to extract the resi-

dues. This technique is more widely used for samples

with low moisture content (cereals) where sample/

analyte binding is more common.

0008For residues that are not suited to multiresidue

extraction methods, dedicated extraction methods

may be used for single or small groups of closely

related pesticides. These methods utilize physical

and chemical properties of analyte and solvent to

carry out selective extraction of the analyte from

matrix. A number of pesticides are normally analyzed

using single residue methods and include formeta-

nate, fluazifop, 2,4-D, formetanate, propamocarb,

and maleic hydrazide.

Clean-up

0009Sample extracts not only contain the target analyte(s)

but may also contain coextractives, such as plant

pigments, proteins and lipids. These coextractives

may have to be removed prior to instrumental analy-

sis to avoid possible contamination of instruments

and to eliminate compounds that interfere during

the determination step. To achieve low detection

limits, sample extracts may also require a concentra-

tion step, which can be incorporated in the clean-up

procedure. Clean-up procedures can lead to losses of

residues and increases in the cost of analysis, and can

reduce the sample throughput. Therefore, a number

of methods utilize minimal clean-up and instead rely

on the selectivity of the detector(s).

0010A number of analytical procedures employ liquid–

liquid extraction for clean-up of sample extracts

by selective partitioning of analytes between two

immiscible solvents. This technique is commonly

used during the analysis of fatty samples. Liquid–

liquid extraction is not easy to automate and requires

the use of large amounts of solvents. (See Analysis of

Food.)

PESTICIDES AND HERBICIDES/Residue Determination 4489

0011 Adsorption chromatography is used in many resi-

due laboratories for the clean-up of sample extracts.

This process involves either:

0012 retention of analyte(s) on a chromatographic

column while the coextractives are unretained:

(the analyte(s) are then selectively eluted from

adsorption medium); or

0013 retention of coextractives on a chromatographic

column while the analyte(s) are unretained.

0014 A number of materials are available for adsorption

chromatography, including alumina, florisil, cellu-

lose, diatomaceous earth (celite), carbon (charcoal,

graphite), silver nitrate, and silica. Alumina and silica

are effective for the clean-up of fatty samples for

organochlorine (OC) pesticide residue analysis. Silver

nitrate is used for the removal of sulfur-containing

intreferences. Carbon has a high affinity for plant

pigments and is particularly suitable for the clean-up

of green leafy vegetable extracts with a high chloro-

phyl content.

0015 Chemically modified silica sorbents are widely

used for the clean-up of sample extracts. These

materials are prepared by the reaction of silanol

groups on silica surfaces with silane reagents to

form esters containing required functional groups.

These sorbents are used in cartridges, disks, mem-

brane filters, and impregnated fiber tips. Some

chemically modified silica and other sorbents used

for clean-up of sample extract are given in Table 3.

0016 Gel-permeation chromatography (GPC) or size-

exclusion chromatography separates molecules on

the basis of their molecular size. Large molecules

(e.g., lipids, pigments, and polymeric coextractives)

elute faster than smaller molecules such as pesticide

residues. The reproducibility, suitability to automa-

tion, and compatibility with a wide range of pesticide/

matrix combinations make GPC a popular clean-up

method in many laboratories worldwide. The disad-

vantages of the technique include the use of large

quantities of solvents, limited sample throughput,

and incomplete separation of high-molecular-mass

pesticides from coextractives. (See Chromatography:

Principles.)

Chromatography and Determination

0017The final stage of the pesticide residue analysis pro-

cedures involves the chromatographic separation and

instrumental determination. Where chromatographic

properties of some pesticides are affected by sample

matrix, calibration solutions should be prepared in

sample matrix. The choice of instrument depends on

the physiochemical properties of the pesticide(s) and

the sensitivity required. As the majority of pesticides

are relatively volatile, gas chromatography (GC) has

proved to be an excellent technique for pesticide

determination and is by far the most widely used. A

typical chromatogram from a multiresidue method is

shown in Figure 1.(See Chromatography: Gas Chro-

matography.)

0018Most residue methods employ splitless injection of

1–3 ml of the sample extract. Cold on-column injec-

tion is employed when the pesticides are likely to

breakdown in a hot injection port. A wide range of

GC column types are used for residue analysis, and

the choice depends on the physicochemical properties

of the analytes. Fused silica capillary columns are

most widely used during analysis of pesticides (see

Table 4). Typical capillary columns are 25–30 m

long with an internal diameter range of 0.1–0.5 mm

and a stationary phase of 0.1–10 mm thickness. Non-

polar stationary phases are used for the separation of

nonpolar pesticide residues such as OC and pyreth-

roids. Similarly, more polar pesticide residues (e.g.,

methadimophos) are separated on relatively polar

columns. The conventional semiselective detectors

are widely used for residue analysis. Electron-capture

detectors (ECDs) are utilized for halogenated pesti-

cides (OCs and pyrethroids). Nitrogen phosphorus

detectors are used for organophosphorus (OP) and

nitrogen-containing pesticides. Flame-photometric

detectors (FPDs) are used for OP and sulfur-

containing pesticides, while atomic emission detect-

ors can be used for a wide range of pesticides.

0019Gas chromatography–mass spectrometry (GC–

MS) has been the predominant technique for the con-

firmation of pesticide residues in the past. Relatively

inexpensive bench-top instruments have made the

technique more widely available for routine screening

in recent years. The resolving power of GC coupled

with the specificity of mass spectroscopy provides the

most effective means of pesticide residue analysis.

A number of ionization techniques are available for

GC–MS instruments and include electron impact (EI)

and chemical ionization (CI). EI impact ionization

tbl0003 Table 3 Examples of sorbents used for clean-up of sample

extracts

Nonpolar Polar Ion exchange

Octadecyl (C18) Florisil SCX

benzenesulfonylpropyl

Octyl (C8) Diol (2OH) PRS sulfonylpropyl

Ethyl (C2) NH

aminopropyl Water’s Oasis

divinylbenzene:

vinylpyrrolidione

copolymer

Polypropylene Silica SAX

trimethylaminopropyl

4490 PESTICIDES AND HERBICIDES/Residue Determination

9.79

11.36

12.98

14.43

16.48

17.45

21.02

21.21

22.14

22.21

22.86

23.42

23.92

24.25

24.49

24.97

26.01

26.33

27.85

29.22

30.08

22.53

fig0001 Figure 1 Chromatogram (GC–FPD) obtained from an injection of cauliflower matrix matched calibration solution containing 0.3 ng of each of following pesticides (and their retention times

in minutes): heptenophos (9.79), ethoprophos (11.36), monocrotophos (12.98), dimethoate (14.43), fonofos (16.48), diazinon (17.45), parathion-methyl (21.02), malaoxon (21.21), fenitrothion

(22.14), pirimiphos-methyl (22.21), malathion (22.53), parathion (22.86), pirimiphos-ethyl (23.42), mecarbam (23.92), methidathion (24.25), tetrachlorvinphos (24.49), profenofos (24.97), ethion

(26.01), triazophos (26.33), pyridaphenthion (27.85), azinphos-methyl (29.22), and pyrazophos (30.08).

leads to a greater degree of fragmentation of molecules

compared with CI. Hence, CI provides a greater sensi-

tivity, but EI provides more spectral information. The

detection systems most widely utilized in residues an-

alysis are based on either the quadrupole or ion-trap

principle. The quadrupole instruments have limited

sensitivity in the scan mode compared with the ion-

trap instruments. However, by operating in the

selected ion mode, adequate sensitivity can be

achieved on quadrupole detectors. A small quadru-

pole mass selective detector can typically detect over

100 pesticides in food extracts at the relatively high

levels (typically 0.2 mg kg

1

) using full scan spectra,

reducing to lower levels (typically 0.01 mg kg

1

) in the

selected ion mode. The ion-trap detectors have a

higher inherent sensitivity, and this allows screening

of clean sample extracts for a wide range of pesticides

at low levels (typically 0.05 mg kg

1

) in full scan

mode. The major advantage of the ion-trap instru-

ments is that the characteristic ions can be selected

and then further fragmented to provide added specifi-

city (MS–MS). (See Chromatography: Combined

Chromatography and Mass Spectrometry.)

0020 High-performance liquid chromatography (HPLC)

is increasingly being used for the determination of

pesticide residues, as it is especially suited to the

analysis of nonvolatile, polar, and thermally labile

residues that are difficult to analyze using GC. The

resolution achieved on HPLC can be comparatively

low, and therefore, the use of selective detection

systems may be necessary for reliable residue analysis.

Ultraviolet (UV) spectroscopy is the most common

choice for detection of (OP) residues in environmen-

tal samples (e.g., soil, water). Although UV detection

is not a very selective technique, it is commonly used

for screening purposes due to its low cost, simplicity,

and wide application range. Elimination of interfer-

ences and optimized chromatography are essential

prior to detection in order to enhance the selectivity

of UV-based methods. The use of diode array

detectors can further enhance the selectivity of UV-

detection procedures. Fluorescence detection offers a

greater selectivity and sensitivity than UV. Pesticides

with inherent fluorescence include dimethoate,

ethoxyquin, azinphos methyl, phosalone, thiabenda-

zole, and carbendazim. With the exception of thia-

bendazole and carbendazim, this technique is not

widely used in pesticide residue analysis, as methods

based on inherent fluorescence have a poor sensitivity

compared with other methods available. Precolumn

and postcolumn reaction systems can be employed

with HPLC methods, which can help improve the

chromatographic separation and detection of ana-

lytes. A number of pesticides (e.g., N-methyl carba-

mates, glyphosate, and phenylurea herbicides) are

analyzed after derivatization to enable fluorescence

detection. The electrochemical detectors are used for

a number of pesticide residues (e.g., captan) in rela-

tively clean samples.

0021The on-line combination of HPLC and mass spec-

troscopy (HPLC–MS) offers a high sensitivity and

specificity, and its use in the field of pesticide residues

analysis is growing. There are a number of ionization

techniques used to interface HPLC with MS ana-

lyzers, of which the most widely used are electrospray

and atmospheric pressure chemical ionization. The

HPLC–MS methods use soft ionization techniques

which typically produce protonated or deprotonated

pseudomolecular ions. Therefore, the chromato-

graphic data do not provide structural information

except when the ions produced are subjected to

successive fragmentation (MS

). (See Chromatog-

raphy: High-performance Liquid Chromatography.)

Derivatization

0022Some pesticide residues require derivitization to en-

hance the extractability, clean-up or subsequent chro-

matographic resolution and determination steps. For

example, pesticides with hydroxy groups are not

suited to GC analysis, and such an analysis may be

possible only after derivatization to esters. Further-

more, esters of certain functional groups can enhance

the detection process, e.g., pentafluorobenzyl deriva-

tives produce a high response on the ECD.

0023Dithiocarbamate pesticides break down to carbon

disulfide (CS

) during analytical procedures. These

residues are determined after treatment of samples

with acidic tin (II) chloride. Any dithiocarbamate

residues in the sample break down to produce CS

gas, which can be trapped in the reaction chamber. An

aliquot of the gas (headspace) in the reaction chamber

is analyzed for CS

using GC–FPD. Alternatively, the

gas produced can be absorbed into a 2,2,4-trimethyl

tbl0004 Table 4 GC stationary phases commonly used for pesticide

analysis (listed in increasing order of polarity)

Stationary phase Typicaluses

100% methyl silicone (DB1) Nonpolar pesticides

5% phenyl, 95% methyl silicone

(DB-5)

Multiresidue screening

35% phenyl 65% methyl silicone

(DB-35)

EPA method 608

50% phenyl 50% methyl silicone

(DB-17)

Polar organophosphorus

pesticides

14% cyanopropylphenyl 86%

methyl silicone (DB1701)

Organochlorine pesticides

50% cyanopropylphenyl 50%

methyl silicone (DB-225)

Polar pesticides

Poly(ethylene) glycol (DB-wax) Polar pesticides

4492 PESTICIDES AND HERBICIDES/Residue Determination

pentane, and an aliquot of the liquid layer is then

analyzed using GC. This approach is more robust

and more amenable to GC–MS analysis compared

with the headspace procedure.

0024 Some pesticides containing sulfur may oxidize to

form sulfoxide and sulfone derivatives before or

during analysis. These products are also toxic and

are included in the residue definition for the monitor-

ing purposes. These residues are analyzed after com-

plete conversion of the pesticide and its sulfoxide to

the corresponding sulfone and thus enable combined

measurement of the pesticide, its sulfoxide, and

sulfone residues. The conversion step involves the

treatment of sample extracts with potassium perman-

ganate in the presence of 2-methyl propan-2-ol. The

sulfone formed is then extracted into an organic

solvent and analyzed by GC.

Other Techniques

0025 Enzyme-linked immunosorbent assay (ELISA)

methods are used for rapid screening of an individual

or a group of closely related pesticides. These

methods require little or no sample clean-up, require

no expensive instrumentation, and are suitable for

field use. ELISA methods are especially suitable

for residue analyses that are not possible using

multiresidue methods. ELISA kits are available for

a number of pesticides, including 2,4-D, aldicarb,

carbendazim, thiabendazole, chlopyrifos, diazinon,

endosulfan, and metalaxyl.

Confirmation

0026 For regulatory purposes, it is essential that pesticide

residues be unequivocally confirmed using MS. How-

ever, if an MS method is not available, the sample

extract is reanalyzed using a different chromato-

graphic column and/or a different detection system

to confirm the initial results.

Emerging Techniques

0027 There are continued improvements in the design of

instruments available for residue analysis. The use

of GC injectors capable of injecting large volumes

can enable determination at low levels. The use of

fast GC, which utilizes an improved design for

heating the columns, can enable faster chromato-

graphic runs and thus enable quicker analysis. Im-

provements in GC instrumentation have enabled

precise control over temperature and gas flow rate.

The use of electronic pressure control devices can

enable more reproducible chromatographic runs,

thus improving the quality of data. These advances,

coupled with more sophisticated software, can enable

more reproducible chromatography, with a typical

retention time variation of 0.01 s. Improvements in

MS instruments will continue to enhance the selectiv-

ity of methods.

See also: Chromatography: Principles; High-

performance Liquid Chromatography; Gas

Chromatography; Mass Spectrometry: Principles and

Instrumentation; Applications; Pesticides and

Herbicides: Types of Pesticide; Types, Uses, and

Determination of Herbicides; Toxicology