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8
Analysis of Organophosphorus Insecticides in Foods

Andres Perez Parada, Lucia Pareja, Silvina Niell, Natalia Besil, Marcos Colazzo,
Maria Verónica Cesio, and Horacio Heinzen

Contents
8.1 Organophosphorus Insecticides: General Overview.................................................................................................................. 181
8.2 Physicochemical Properties........................................................................................................................................................ 182
8.3 Maximum Residue Levels........................................................................................................................................................... 184
8.4 Analysis of OP Pesticides........................................................................................................................................................... 185
8.5 Sample Preparation..................................................................................................................................................................... 186
8.6 Sample Preparation Protocols for the Food Analysis of Organophosphates.............................................................................. 186
8.7 Sample Preparation Methods...................................................................................................................................................... 187
8.8 Luke and Mini-Luke Methods.................................................................................................................................................... 187
8.9 Ethyl Acetate Method................................................................................................................................................................. 187
8.10 Quick, Easy, Cheap, Effective, Rugged, and Safe...................................................................................................................... 188
8.11 Matrix Solid-Phase Dispersion................................................................................................................................................... 189
8.11.1 Microwave-Assisted Extraction.....................................................................................................................................190
8.11.1.1 Solid-Phase Microextraction..........................................................................................................................192
8.12 Instrumental Determination of Organophosphate Pesticide Residues in Foods........................................................................194
8.12.1 Thin-Layer Chromatography..........................................................................................................................................194
8.13 Gas Chromatography..................................................................................................................................................................194
8.13.1 Conventional Detectors for OPs.....................................................................................................................................194
8.13.1.1 Flame Photometric Detector...........................................................................................................................194
8.13.1.2 Nitrogen–Phosphorus Detector.......................................................................................................................194
8.13.1.3 Electron Capture Detector..............................................................................................................................194
8.14 Mass Spectrometric Determination of Pesticide Residues.........................................................................................................194
8.14.1 Gas Chromatography–Mass Spectrometry.................................................................................................................... 195
8.15 Liquid Chromatography/Mass Spectrometry............................................................................................................................. 195
8.16 Liquid Chromatography Coupled to MS/MS.............................................................................................................................197
8.17 Conclusions.................................................................................................................................................................................197
References............................................................................................................................................................................................. 198

continuous process that allows the nerve impulse to be transmit-

8.1 Organophosphorus Insecticides: General
Overview
There are roughly 170 insecticidal organophosphorus (OP) com-
pounds belonging to different chemical families. This chapter
will deal with the analytical aspect of these compounds. Other
OP compounds such as glyphosate will not be discussed, as it has
totally different biological properties and it deserves a review for
itself alone.
Organophosphate insecticides are one of the most employed
crop protection classes of compounds. First introduced in the half
of the past century, they are active at the interspaces of the neu-
ronal junction through the inhibition of the acetyl cholinesterase,
the enzyme responsible for the recycling of the neurotransmitter
acetylcholine and the repolarization of the neuronal membrane, a
ted throughout the body of animals and insects (Cremlyn 1992).
From a general chemical point of view, they are organic esters
of phosphate and thiophosphate with a specific substitution pat-
tern. They bear an electrophilic leaving group that is lost when
the phosphate is linked to the hydroxyl group of the serine place
at the active site of the enzyme. The O–P bond thus formed is a
very stable one, hampering the regeneration of the free enzyme
(Figure 8.1).
R = alkyl, alkylthio, amino substituted, and so on. Y is a good
leaving group, and X = S,O.
These compounds exhibit a relatively high toxicity toward
all the organisms that relay in the acetyl choline cycle for their
living but on the other hand, they do not accumulate in the
environment as other pesticides such as the organochlorines
(OCs), where they are easily degraded by soil microbiota

181
 182
RO X Although lipophilic compounds, the polarity of organophos-
P phates is an intermediate one. If a pesticide residue determi-
O Y
Figure 8.1  General chemical structure of OP insecticides.
(Tilak et  al. 2005). Although the major toxicological con-
cern is focused in the acute casualties caused by organo-
phosphates, the continuous exposure to these chemicals had
shown to cause the dysfunction of many acetyl choline-medi-
ated processes that lead to neurodegenerative illness such as
Parkinson’s disease (Misik et al. 2014; Wang et al. 2014). Most
of these compounds are classified to have a toxicity level of
class I, II, and in the best cases as class III and for that reason,
many of them are no longer in use, after decades of extensive
application over the produce (Shah and Iqbal 2010).
Nevertheless, they are cheap compounds that are still widely used
in most countries, mainly by the food producers from Asia, Latin
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015
America, and Africa. The new insecticides such as the neonicoti-
noids are much more expensive and OPs do not suffer from a global
insect resistance phenomena such as the pyrethroids, although there
are a number of reports on the increasing resistance developed by
insects toward these compounds (Aïzoun et al. 2013). Figure 8.2
shows some representative examples of organophosphates.
8.2  Physicochemical Properties
The specificity of OPs action toward the different insect families
as well as the possible harm to the environment is governed by
the particular molecular composition of each compound that can
be globally understood through the physicochemical properties
that rule not only the pharmacokinetics but also their toxic action.
Organophosphate insecticides are lipophilic compounds, a prop-
erty that is the key feature for their mode of action. They have to
diffuse into the lipophilic myelin sheath to reach their target. The
property that explains this behavior is the differential solubility
they have toward the aqueous internal environment of the body
and the membrane lipids (Zhao and Yu 2013). Most of the OPs
are sparingly soluble in water but are soluble in common organic
solvents. The quantitative relationship between these solubili-
ties is established through the partition coefficient, named Kow,
a constant that shows the distribution of a compound between an
organic and an aqueous phase. The Kow allows the prediction and
understanding of the distribution of these as in compound into
the different body compartments. The Kow is also the most useful
physicochemical property to study the extractability of a pesticide
residue from its matrix when evaluating the suitability of a sam-
ple preparation procedure for OP residue determination. Usually,
food matrices have roughly 80% water content and therefore, the
pesticide partitions between the extraction solvent and the water
belonging to the matrix. Kow is the relationship between the con-
centration of the compound in the organic phase and the concen-
tration of the compound in the aqueous phase (Cremlyn 1992).
Kow = [C]octanol/[C]aqueous phase
A transformation that turns the Kow in a handful expression is
the –logP, and a lipophilicity scale can be built out (Table 8.1).
Handbook of Food Analysis
nation has to be performed in an oily matrix or one that has a
relatively high proportion of fatty compounds, such as edible
oils, nuts, avocado, butter, or fatty meat, they propose an ana-
lytical challenge. If an apolar solvent such as hexane is used for
extraction, fats will be extracted along with the pesticides and
further cleanup steps have to be performed to eliminate triglyc-
erides from the extract. Fats contaminate the chromatographic
systems, particularly in gas chromatography (GC) determination
as they not only poison notably the electron capture detectors
(ECDs) but also spoil liners in the injector systems where they
slowly degrade, rising up the noise at the detector (Li et al. 2014).
A possible sample treatment strategy could be then, to extract the
sample with a polar solvent where fats are sparingly soluble, such
as acetonitrile (MeCN), methanol, or ethanol. Nevertheless, dur-
ing the extraction step with these relatively polar solvents, the less
polar pesticides, for example, parathion methyl, or ethion, will par-
titionate between fats, usually triglycerides and the solvent itself.
These strategies are successful to yield quantitative recoveries only
if the solvent polarity is diminished using solvent blends, typically
MeCN saturated with hexane (Pérez et al. 2010). Another proven
procedure to eliminate fats from pesticide extracts for residue anal-
ysis is to reextract the organic solution with hexane and the lipids
are extracted in the hexane layer (Niell 2014) but, the most common
procedures are to apply a chromatographic separation either using
a normal polar-stationary phase (Florisil, silica gel, and alumina)
like in the Luke method (Luke et al. 1981) or through reverse-phase
(RP) chromatography, typically C8 or C18-modified silica gel. As
the MeCN extract is passed through an RP cartridge, the more
polar compounds are not retained but the MeCN has not enough
strength as the eluting solvent, so the fats are retained in the non-
polar phase. The other chemical difference that permits the separa-
tion of fats from trace amounts of pesticides is the molecular size.
Triglycerides normally have a molecular weight (MW) of 700 g/
mol, whereas the MW of pesticides is around 200–400 g/mol. Size-
exclusion chromatography offers the possibility of performing such
separations. The high MW fats elute first and the pesticides elute
at a higher elution volume. The procedure can be automated and
the productivity of the whole procedure is enhanced. Nevertheless,
normal fats and oil constituents such as sterols, cholesterol, and the
phytosterols, have MW of around 380–400 g/mol and are difficult
to separate from high MW pesticides (Pérez et al. 2010).
Parathion, malathion and their transformation products (TPs),
the oxons, as well as chlorpyrifos are typical lipophilic compounds
that are easily extracted from the aqueous matrices with almost
any organic solvent. Some OPs, such as acephate and dimethoate
are more polar, so the partition process in aqueous media, would
yield low recoveries if additional precautions such as the salting
out of the extraction media are not undertaken (Agüera 2004).
Metamidofos, for example, is water soluble and can also stand as
an acephate TP, as well as the oxidation of dimethoate yields the
more soluble TP, omethoate (Footprint 2006).
Oxidation of phosphothiones usually yields the more toxic but
also hydrolysis-sensitive corresponding oxon (see Equation 8.1).
RO S RO O
P CytochromeP450
P (8.1)
O Y RO Y
 Analysis of Organophosphorus Insecticides in Foods 183

O S O
O O O O O
P O P P
O P S S
O O O S
N Ethion
CI O H

Monocrotophos
O O
H
CI CI O O

S
Chlorfenvinphos
O S Malathion
S
CI P
CI P
O S
Cadusafos O
O
S
O O H
O CI N
P
P O S
O O O O
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015

Dichlorvos P O
N O O O
S Dimethoate
N Coumaphos
O
N S CI N O
O
P
S P O O
O S
O S
P N O CI CI
O
S Phosmet Chlorpyrifos
Azinphos-methyl
O

Fenthion
Diazinon Acephate
O
O
P O
N O O
O
P S O
N
O S O S NH
P
Parathion S
O P S O
S
O O Propetamphos
S O
P
N Pirimiphos-methyl O
O P O

NH O O
N N

H O
N
O P O
O O O NO2
S
P P
O O
O O Trichlorfon
S
CI
O 2N Fenitrothion HO
Isofenphos CI
CI
O O
O O
O N O
P P P
S
O O
O S S
Br CI CI CI N

Profenofos Dichlofenthion Quinalphos

Figure 8.2  Some of the most representative OP insecticides.

 184 Handbook of Food Analysis

Table 8.1 such as sulfonyl ureas. Microwave-assisted extraction (MAE)

Kow, Octanol/Water Constant, and Henry’s Constant of for OPs have replaced the old sample preparation methods that
Some Important Organophosphorus Insecticides included Soxhlet extractions and nowadays is a common proce-
dure for sample preparation due to shorter extraction times (Niell
Pesticide −logP Henry’s Constant KH et al. 2011). Nevertheless, some OPs such as dichlorvos have a
Acephate −0.85 2.5 × 10−11 vapor pressure of two orders of magnitude higher than chlorpy-
Azinphos methyl 2.96 5.15 × 10−08 rifos or diazinon and can be lost during the sampling preparation
Chlorfenvinphos 3.8 2.20 × 10−08 steps, particularly when evaporating the extraction solvent.
Chlorpirifos 4.7 2.80 × 10−04
Chlorpirifos methyl 4.00 1.91 × 10−04
Cadusafos 3.85 5.42 × 10−05
Diazinon 3.69 6.1 × 10−02
8.3  Maximum Residue Levels
Dimethoate 0.704 4.10 × 10−08 For all the substances applied on the produce, Food and
Ethion 5.07 1.58 × 10−05 Agriculture Organization (FAO)/World Trade Organization
Fenchlorphos 4.88 3.3 × 10−07 (WHO) Joint Commission through the Codex Alimentarius rules
Fenitrothion 3.32 3 × 10−06 the maximum concentration limits permitted in the different
Fenthion 4.84 1.01 × 10−05
matrices that are not noxious for human health and the environ-
Malathion 2.75 4.8 × 10−05
ment. The Codex Alimentarius is a suprainternational forum of
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015

Metamidophos −0.79 1.6 × 10−06

180 countries, where consensus are always pursued. The agree-
Methidathion 2.57 7.5 × 10−08
Omethoate 0.74
ments reached there, not only ensure the health of the population
2.9 × 10−08
Parathion ethyl 3.83 2.5 × 10−05
but also trade between countries (Codex Alimentarius 2014).
Parathion methyl 3.00 2.3 × 10−06 Setting MRLs is the crossover of two separated roads that has
Phosalone 4.01 5.8 × 10−06 to be considered before a legal definition is taken. On one hand,
Pirimiphos methyl 3.9 3.8 × 10−04 is the amount of the pesticide residue that is left in the commodi-
Propenophos 1.7 1.39 × 10−05 ties for after their application on crops following good agricul-
Pyriproxyfen 5.37 4.74 × 10−06 tural practices (GAPs) and on the other, are the toxicological
Tetradifon 4.67 1.46 × 10−08 properties of the chemical under study. After toxicological stud-
ies on different animal species, parameters such as lethal dose
Source: Data gathered from FOOTPRINT. 2006. The
50 (LD50), which show the potential risk for an acute intoxi-
FOOTPRINT Pesticide Properties DataBase. Database
collated by the University of Hertfordshire as part of the cation in a single intake, the no adverse effect level (NOAEL)
EU-funded FOOTPRINT project (FP6-SSP-022704). that is a guide to determine a level where no toxic effect is
http://www.eu-footprint.org/ppdb.html. observed after intake, and the lowest-observed adverse effect
level (LOAEL), the minimum concentration of a compound at
Oxidations that do not occur around the phosphate portion which an adverse effect is observed, are determined. As these
of the molecule can yield compounds that are also pharmaco- experiments are performed in animal species, extrapolation to
logically active and therefore, their determination is mandatory humans has to be performed (Codex Alimentarius 2014). The
(SANCO document No/12571/2013). The most striking example picture is even more complex as special susceptible popula-
is fenthion, whose all TPs are biologically active (Equation 8.2). tion groups such as pregnant women, children, and old people
have to be considered, and therefore, the toxicological values
S obtained from the extrapolation from animals to humans are
H3C S O P OCH3 minimized applying safety factors (SFs). Taking these SFs into
OCH3 account, an acceptable daily intake (ADI), is fixed. The ADI is
H3C
the amount of pesticide expressed in milligram per kilogram
Fenthion of body weight that can be consumed safely by a person during
O S O S his/her entire life. The application of the pesticide of different
H3C S O P OCH3 H3C S O P OCH3 commodities will leave a residue that eventually will be eaten
OCH3
O
OCH3 (8.2) by the population. The participation of each food in the daily
H3C H3C diet of the population and therefore the whole pesticide intake
Fenthion sulfoxide Fenthion sulfone   has to be considered. The total amount of pesticide that could be
ingested after considering as a sum of all the sources where the
Most of the organophosphates have relatively high vapor pesticide is applied has to be lower than the determined ADI for
pressure and the boiling point is below 250°C, also being heat that pesticide. With these figures in hand, an MRL for a pesti-
insensitive. This important property turns organophosphates cide in a specific food is fixed. If the pesticide intake to which
GC-amenable compounds giving a straightforward tool for their the population is exposed is lower than the residue left after the
analysis, either as raw materials or at trace level. Even more, their application to the different commodities, at the recommended
stability toward heat allows the use of microwave radiation to per- dose in the field following GAPs, the pesticide is allowed to be
form very efficient extraction procedures from different matri- in the market. Otherwise, the pesticide is banned. The MRLs
ces, a procedure that is not possible to apply to heat-sensitive fixed according to this procedure should never be higher than
compounds such as pyrethroid insecticides or some herbicides the ADIs for the specific pesticide. The MRLs setting depends
 Analysis of Organophosphorus Insecticides in Foods 185

on the GAPs, and therefore the registration which establishes in Table 8.2
which commodities the pesticide will be used. If the combina- Codex Alimentarius MRLs for OPs in Selected
tion of matrix–pesticide is not considered in the Codex, then the Commodities
presence of the pesticide in such a commodity must be consid-
MRL
ered a violation. In such cases, the MRL is the limit of detection
Commodity Pesticide (mg/kg)
(LOD) of the analytical method (Codex Alimentarius 2014).
Each region or country has its own dietary peculiarities, where Almonds
the intake of a specific food depends many times on tradition, and Azinfos methyl 0.05
therefore, the population exposure to a specific toxicant applied Chlopyrifos 0.05
in the field over a crop, a vegetable, or an animal is different for Diazinon 0.05
people living in East Asia, Europe, or America. Therefore, to set- Methidathion 0.05
tle out the MRLs, these peculiarities must be considered, but for Phosalone 0.1
the MRLs stated in the Codex Alimentarius, the diet that is taken Rice dehulled
for the MRLs Codex calculations is a standard occidental one. Acephate 1
Nevertheless, the most important food markets such as Fenthion 0.05
European Union (EU), United States, Russia, and Japan also have Metamidofos 0.6
their own regulations. The exporting countries to these regions
have to negotiate the MRLs in their exports to be allowed to sell Artichoke
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015

them there. As said above, International Organizations such as Acephate 0.3

European Food Safety Authority (EFSA) in the EU or the Food
and Drug Administration (FDA) in the United States have their
own regulations for pesticides. Particularly, the EU fixed a maxi-
mum residue level (MRL) for those pesticides that are not listed in
the ANNEX I of the resolution Commission Regulation (EU) No
212/2013 for a given combination of commodity–pesticide, at the
0.01 mg/kg concentration level. This drives the mandatory LOD
that pesticide residue laboratories have to determine pesticide
residues below 0.01 mg/kg, which was, at the time the ANNEX I
was approved, the very state of the art in pesticide residue analy-
sis. This is also the case of OP compounds and this level of MRLs
has been settled. Many OPs are now banned and the international
trade, forces the labs in food-importing countries, such as EU,
United States, or Russia, to cover the whole palette of pesticides
that can be found, even those that have been banned for years.
The MRLs are the limit of quantification that has to be reached
to comply with the regulations for all pesticide residue laborato-
ries. In many cases, the MRLs definition is more complex, as in
the already-mentioned malathion/maloxon or fenthion cases, as
it states in Table 8.2.
In such cases, all the metabolites of toxicological significance
have to be determined. They are all summed up and expressed as
the parent compound. In the drinking water-quaility regulations,
no individual pesticides should be at levels higher than 0.1 μg/kg
but the total amount of pesticides should not exceed 0.5 μg/kg.
For example, in the water-quality regulations, no individual
pesticides should be at levels higher than 0.1 μg/kg but the total
amount of pesticides should not exceed the 0.5 μg/kg.
As stated above, a pesticide residue is the amount of active
substance or its degradation products that are present in a spe-
cific commodity after an application of a pesticide following
GAPs. The residue definition for a given substance must there-
fore include those active degradation products. That is the case
for a number of pesticides, for example, parathion, malathion, as
well as the already-mentioned fenthion.
The SANCO document No/12571/2013 gives an example on
how to report the residue of such compounds in a sample. The
total residue is expressed as the concentration in milligram per
kilogram of the pesticide after converting the concentration
found for each degradation product in what it represents from the
Dimethote 0.05
Metamidofos 0.2
Sorghum
Chlorpyrifos 0.5
Chlorpyrifos methyl 10
Malathion 3
Methidathion 0.2
Terbufos 0.01
Source: From Codex Alimentarius. 2014. http://
www.codexalimentarius.org.
original active principle. As an example, in the case of convert-
ing fenthion sulfoxide (SO) into fenthion, this is expressed as
CFenthion SO to Fenthion = MwFenthion/MwFenthion SO × CFenthion SO
= 278.3/294.3 × C Fenthion SO = 0.946 × C Fenthion SO
The calculation is repeated for each metabolite and the conver-
sion factor, which is the ratio between the MW of fenthion and
the MW of each TP is multiplied by the concentration found for
each TP under study. Finally, all the equivalences of TP to fen-
thion and the amount of fenthion found are summed up to yield
the total fenthion residue that has to be reported.
CFenthion Sum = 1.00 × CFenthion + 0.946 × CFenthion SO
+ 0.897 × CFenthion SO2 + 1.06 × CFenthion oxon + 1.00
× CFenthion oxon SO + 0.946 × CFenthion oxon SO2
Organophosphates with complex residue definition are shown
in Figure 8.3.
8.4  Analysis of OP Pesticides
There has been a multitude of reports on method development for the
determination of OP residues. Many of them are of academic inter-
est or miscellaneous ones, but their application to everyday situation
is doubtful. Actually, a real alphabet soup to describe these meth-
ods exists, such as accelerated solvent extraction (ASE), dynamic
liquid–liquid (DLL), dimethyl ether extraction (DEE), matrix solid
 186 Handbook of Food Analysis

O O O O CH3
H3C P
S O P OCH3 O S
O O H3C S
O OCH3
O P
H3C S P CH3
O O O O
O O CH
3
Fenthion S

Malathion Parathion Phosmet

O O NO2 S
S S O
P S P O
O
O
S O P O N
O O
O O O

O NO2 O O
O
C S OCH3 S P O
O P
O OCH3 H3C O P O N O
O C O
O H3C O
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015

Figure 8.3  Examples of organophosphates and their toxicological relevant metabolites.

phase (MSP), and microwave assisted extraction (MAE) among oth-
ers. In this chapter, we will focus on the modern methods of OPs
analysis that could be implemented and performed in routine labo-
ratories devoted to pesticides residue analysis in food.
8.5  Sample Preparation
In any analytical method but particularly those of trace com-
pounds, the sample preparation steps and the instrumental deter-
mination have their own particularities that have to be considered.
8.6 Sample Preparation Protocols for the Food
Analysis of Organophosphates
The new concepts on pesticides residue analysis and the appli-
cation of the last-generation mass spectrometer coupled either
to liquid chromatography or gas chromatograph (LC, GCs) have
dramatically changed the pesticide analysis in foods and conse-
quently, organophosphate residue analysis in the last 10 years.
Pesticide residue analysis nowadays is focused on the develop-
ing of environmental-friendly procedures, seeking the minimi-
zation of the reagent consumption and waste disposal. Following
this goal, the greater the number of analytes that can be deter-
mined in a single analytical step, the better. This idea drove to
the development of the multiresidue analysis concept, where a
high number, typically more than 20 pesticide residues belonging
to different chemical families, can be determined in a single ana-
lytical procedure. This concept was presented for the first time in
the late 1980s but the limitations in the instrumental sensitivities
hampered the full development of this type of procedure.
Nevertheless, most analytical procedures were developed to
perform a single sample preparation step. The resulting extract
was then splitted in the number of portions needed to perform the
different chromatographic determinations when possible (VWA
1994) (Figure 8.4).
Actually, the advent of modern instrumentation based on
high sensitivity determination of analytes through tandem mass

Sampling

Homogenization

1. N-methylcarbamates
Extraction 2. Benzimidazole fungicides
3. Benzoylureas
4. Phenylureas
Cleanup
GC SPE1, SPE2... 5. Prochloraz, Imazalil

HPLC

ITD ECD FPD NPD

1 2 3 4 5

Figure 8.4  Analysis scheme for multiresidue methods. (From A.R. Fernandez-Alba, personal communication, Santa Fe, 2006.)
 Analysis of Organophosphorus Insecticides in Foods
spectroscopic methods after their chromatographic separation,
either through LC or GC, expanded the scope of the multi resi-
due methods (MRMs) of analysis. Within this context, analytical
methods claiming to analyze up to 500 pesticide residues have been
developed. Nevertheless, there are still some reports on the mul-
tiresidue analysis of organophosphate-class compounds based on
the use of selective and specific GC detectors, such as FPD (flame
phosphorus detector) and NPD (nitrogen and phosphorus detector).
The earlier 2004 SANCO guidelines accepted determinations
done using selective and/or specific detectors that are further
confirmed by an independent method, for example, GC–MS (gas
chromatography–mass spectrometer). But this document since its
2011 edition, does not permit this possibility. Therefore, within
the official laboratories in the EU, the only official analysis rec-
ognized is the determination of pesticide residues through mass
spectrometric measurements except for a bunch of pesticides, but
this rule does not apply for organophosphates. Nevertheless, the
use of conventional detectors has wide application in other parts
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of the world. The FDA still accepts the determination of OPs
through conventional GC detectors (FDA 2014).
The modern methods for analysis of organophosphate pesti-
cides in foods are included in these methods and will be dis-
cussed accordingly. The high sensitivity of MS detectors drove
the change in the sample preparation procedures for pesticide
MRMs of analysis. Samples are miniaturized; the number of
sample preparation steps has been minimized as well as the
organic solvent consumption.
8.7  Sample Preparation Methods
The sample preparation methods for routine pesticide residue
analysis are actually procedures focused on the simultaneous
determination of insecticides, fungicides, and herbicides. The
performance of these methods will be discussed regarding how
they adjust for the OPs analysis.
8.8  Luke and Mini-Luke Methods
The Luke method (Luke et  al. 1981) first introduced in the
1970s has been one of the most popular multiresidue methods
for pesticides residue analysis. It has been specifically designed
for high-water-content matrices but since its introduction, many
modifications were assayed to apply it to other different matrices.
The method starts as shown in Figure 8.5, with an acetone
extraction of the sample. Acetone is a water-miscible solvent that
forms a continuous liquid phase that extracts the pesticide from
the matrix. The liquid phase is then extracted with a mixture of
dichloromethane (DCM)–hexane (1:1). Lipids and pesticides are
extracted with the apolar phase which, in the old Luke method,
was chromatographed through a Florisil column. The mini-Luke
method takes advantage of the advances in the instrumentation
and the column chromatographic step is no longer performed
(WMA 1994). The Luke method is suitable for most organo-
phosphate pesticides, particularly the most apolar ones, such as
ethion, and parathion but the very polar ones, such as dimetho-
ate/omethoate as well as acephate/methamidophos could be lost
during the extraction step.
187
Fruit/vegetable
+ Acetone/water
Agitation/filtration
Extraction
solution
Organic phase I a
+ Aqueous solution
Organic phase II
NaCl/DCM
DCM Fats + Organochlorines
Chromatographic AcOEt Organophosphates
column (florisil) + Pyrethroids
AcOEt/ Most polar
MeOH compounds
(herbicides)
Figure 8.5  Luke method.
Nowadays, the whole procedure has been miniaturized and
the extraction and mixing step is performed with a high-speed
Ultraturrax. An aliquot is taken, sodium chloride is added, and
extracted with DCM:hexane. The solution is evaporated, volume
is adjusted, and injected in the chromatograph. The whole proce-
dure is straightforward and useful.
8.9  Ethyl Acetate Method
Pesticide residue extraction using ethyl acetate as the solvent,
is also one of the most employed methods for sample prepara-
tion for pesticide residue analysis (Pihlström et al. 2007). Ethyl
acetate (EtOAc) as a solvent has unique properties, such as the
possibility of dissolving polar and apolar compounds with equal
efficiency. Even the extraction of lipophilic pesticides from fat-
rich matrices with water-miscible solvents such as MeCN or
acetone is sometimes not easily performed, and can be quan-
titatively achieved with EtOAc as lipids are easily dissolved in
it, together with the searched pesticides. Fats are then further
eliminated using, for example, GPC (gel permeation chromatog-
raphy). The gas expansion volume of EtOAc makes it a useful
option for large volume injection (LVI) in GC. The noninterfer-
ence with any of the GC (FPD, NPD, ECD, and MS) detectors
is another of its advantages, allowing the direct injection of the
extract without performing a solvent change. This advantage in
GC analysis turns into a drawback when LC determinations have
to be performed. The extracts have to be redissolved in methanol
or MeCN. Although it is a routine procedure, losses had been
reported when changing the solvent to a more polar one if lipidic
matrices are under analysis (Agüera 2004).
There are three actual miniaturized procedures that are
modifications of the original method as proposed by Swedish
 188 Handbook of Food Analysis

Fruit and vegetables Cereals Animal origin, A Animal origin, B

Extraction Extraction Extraction

Ethyl acetate Ethyl acetate Ethyl acetate
5 g sample Fat determination
5 g sample 5 g sample
falcon tube falcon tube falcon tube

Centrifugation Centrifugation Filtration Extraction

EtoAc+CH

Centrifugation Filtration

Purification
GPC, SX-3
Filtration Filtration
Centrifugation
Inject to GC- Inject to
Direct Direct Direct Direct MS/MS UPLC-MS/MS Evaporation
inject inject inject inject
to GC- to UPLC- to GC- to LC-
MS/MS MS/MS MS/MS MS/MS Reconstitution
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015

Inject to
Inject to
UPLC-
GC-MS/MS
MS/MS

Figure 8.6  Application of SweEt method to the different food matrices. (From S. Ekroth, personal communication, LAPRW, 2011.)

workers. The method has been adapted in Sweden to meet the
requirements of the new trends in pesticide residue analysis,
named SweET (Pihlström et al. 2007). The matrix is extracted
with EtOAc buffered with sodium bicarbonate. The procedure is
routinely carried out by the National Food Authority in Sweden
(Figure 8.6).
Mol et al. (2007) also introduced a variation of the traditional
EtOAc method by including a dispersive cleanup step based on
the success of the QuEChERS (quick, easy, cheap, effective, rug-
ged, and safe) method. The extract was treated with GCB (graph-
itized carbon black) to remove pigments and PSA (primary and
secondary amine) to neutralize acidic compounds. The latter was
more effective in removing coextractives that appear either in
the GC or LC chromatogram, whereas the former could success-
fully remove all the chlorophyll when EtOAc was used solely as
the solvent. Blends with aromatic solvents such as toluene and
xylene performed poorer clean ups but the losses of pesticides
such as azinphos methyl, thiophanate methyl, and chlorpyrifos
methyl among others were minimized. The amounts of GCB and
PSA were optimized and it could be stated that significant losses
of pesticides were observed using 200 mg/L of PSA (Table 8.3).
Table 8.3
Losses Registered by Dispersive Cleanup Using
a = 25 mg/L and b = 200 mg/L PSA in Ethyl Acetate
OPs Recovery (%)
Acephate 43a
Dichlorvos 33a
Dimethoate 62b
Mevinphos 62b
Phosphamidon 63b
Profenofos 56b
A further simplification of the EtOAc method was proposed
by Ferrer et al. (2010) who use no cleanup step and the extraction
solution, after salting out and drying are directly injected in the
system. Nevertheless, the higher load of coextractives associated
with this method can yield lower values in liquid chromatogra-
phy coupled to MS/MS (LC-MS/MS), due to ion suppression,
as observed in the recent interlaboratory test, IMP-37, organized
by the European Institute for Reference Materials (ERM) (ERM
2014), where all the participating laboratories that used EtOAc
extraction yielded lower results, as they did not correct for recov-
ery following the SANCO guidelines. The EtOAc method is an
excellent option for the GC analysis of pesticide residues, as no
solvent change has to be performed. A QuEChERS adaptation
has been reported that uses EtOAc as the extraction solvent but
for conventional GC detectors. The reagents and sample amounts
were adjusted; 60 g of sample are used instead of the usual 10 g
used when mass spectrometers are used for pesticide residue
determination (Aysal et al. 2007).
8.10 Quick, Easy, Cheap, Effective,
Rugged, and Safe
The acronym QuEChERS stands for quick, easy cheap, effec-
tive, rugged, and safe. The name was coined by the method
inventors, Steven Lehotay and Michelangelo Anastassiades in
2003 (Anastassiades 2003). After a few years of its introduction,
it has become the most popular sample preparation method for
pesticides residue analysis. It is a development of the old MeCN
method introduced by Mills in the 1960s. It is a template that,
supported by a number of milestones, can be adapted for almost
any matrix. The method is thought for the MS determination of
the residues, taking advantage of the sensitivity of the new mass
spectrometers. Nevertheless there are some applications using
 Analysis of Organophosphorus Insecticides in Foods
10 g sample + 10 mL MeCN
ut
i n g-o n
lt io
Sa trat 4 g MgSO4 + 1 g NaCl
ex vigorous agitation 1-4 min
Centrifuge 1 min at 3400 rpm
ive
ers
d isp -up 1 mL supernatant + 25 mg PSA + 150
E n
SP clea mg MgSO4, agitate 20 s (VORTEX) &
centrifuge1 min a 3400 rpm
Figure 8.7  Schematic representation of the original QuEChERS
method. (From Anastasiades, 2003.)
conventional GC detectors, as mentioned above. QuEChERS is
a miniaturized method where the amount of sample and solvent
stands to yield a solution of 1 g/mL. After the salting out step,
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015
MgSO4 is aaded to the organic phase to remove the remains of
water. An aliquot of the acetronitrile phase is then submitted to a
straightforward dispersive cleanup step with PSA to retain acidic
compounds, dried with MgSO4 and can be directly injected in the
LC. In case that GC analysis has to be performed, MeCN is evap-
orated and the residue is redissolved in isooctane and injected.
This basic procedure can be modified in various ways, depend-
ing on the type of matrix to be analyzed. PSA can be avoided if
acidic pesticides are searched for. In the case of lipid-rich matri-
ces, RP C18 silica can be added. MeCN as the solvent is not strong
enough to desorb lipids from the RP C18. Therefore, lipids are
removed from the solution and cleaner extracts can be injected in
the chromatograph (QuEChERS 2014).
The same objective can be accomplished taking advantage of
the immiscibility between hexane and MeCN. After the separa-
tion of the organic and the aqueous phases, hexane can be added.
Lipids are extracted to the hexane phase, cleaning up the pesticide-
containing solution. Many pesticides are weak bases and when
acid matrices such as citrus fruits are analyzed, these pesticides
can be lost during the extraction procedure. To skip this problem,
the extracting solution should be buffered. There are two  main
Matrix
Sorbent
Figure 8.8  Schematic flux for MSPD.
189
methods, based on QuEChERS template, one is the official
method AOAC, 2007.1 that uses sodium acetate to stabilize the
pH to pH = 65. Further on, European (CEN) method stabilizes the
pH through the addition of citrate salts (Anastassiades et al. 2007).
The three methods thus described are used without problems in
the sample preparation for the analysis of OP pesticides. A specific
report of QuEChERS application to analyze 98 OPs and carba-
mates (Chung and Chan 2010) has been published (Figure 8.7).
8.11  Matrix Solid-Phase Dispersion
Matrix solid-phase dispersion (MSPD) is a procedure usually
applied for sample treatment in a variety of matrices. This pro-
cedure is based on the mechanical disruption of the sample in
a proper sorbent material with a mortar and a pestle. Sample
homogenization, extraction, and cleanup can be accomplished
simultaneously by using a relatively small sample size, low sol-
vent consumption, and minimum amount of sorbent phase. After
blending, the sorbent material is packed into a column and the
analytes are eluted using a suitable eluting solvent. However, the
selection of the experimental conditions is critical for the selec-
tive extraction and purification of the sample extract (Barker
2007) (Figure 8.8).
Many MSPD procedures are focused on the combination of
cocolumns, which is the use of a packed sorbent with complemen-
tary features of the disrupting phase at the bottom of the column
to obtain exhaustive removal of interferences. Hence, the selec-
tion of the proper dispersant phase plus the elution volume is man-
datory to achieve enhanced extraction of the matrix while giving
purified extracts for quantitative analysis of pesticides. The use of
cocolumns has improved the applicability of MSPD by increasing
the versatility of the purification step (Barker 2007).
MSPD is currently used for extracting analytes from both solid
and liquid samples. The potential of this strategy is focused on
troublesome matrices such as propolis. The appropriate selection
of conditions for enhanced extraction of polyphenols in propolis
tinctures was recently described (Pérez-Parada et  al. 2011) for
the analysis of ethion, chlorpyrifos, and coumaphos in propolis
0 1 2 3 4 5 6
Tempo (nm)
 190
tinctures. Samples were dispersed onto anhydrous Al2(SO4)3
using an MSPD approach with Florisil cocolumn cleanup by per-
forming elution with CH2Cl2:EtOAc (9:1) solvent with GC–FPD
and GC–MS determination. LODs were below 26.0 μg/kg in
GC–FPD and 1.43 μg/kg for GC–MS analysis. Mean recoveries
were in the range of 85%–123% with relative standard deviation
(RSD) values below 13%.
On the other hand, various authors have used MSPD for the
analysis of OP residues in medicinal plants. Zuin et  al. (2003)
reported the determination of four OPs in Passiflora L. leaves
using an MSPD on Florisil with an Na2SO4 cocolumn to remove
wet leaves by eluting with an n-hexane:EtOAc (7:1) mixture and
GC–ECD determination. Acephate was included in the scope of
a multiresidue method for Cordia salicifolia leaves by using neu-
tral alumina and peat as dispersant phases plus Na2SO4 and C18
cocolumns and eluting with cyclohexane: CH2Cl2 mixture and
GC–MS determination (de Carvalho 2009, 2010).
A miniaturized online MSPD–LC–DAD (diode array detec-
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tion) method for the analysis of nine OPs was reported (Valencia
and García de Llasera 2011): 50 mg bovine tissue was dispersed
in 200 mg of C18 and packed at the head of a stainless-steel car-
tridge containing 50 mg silica as the cosorbent. The cartridge
was then placed in an online assembly, with a C18 precolumn and
column. The cartridge was washed with water and water/MeCN,
and the analytes of interest eluted with MeCN in a dynamic mix-
ing chamber, where MeCN was diluted with water and loaded
into the precolumn for analysis by using gradient elution and
ultraviolet (UV)–DAD detection. Recoveries ranged from 91%
to 101% with RSD ≤12% with LODs in the 0.5–1 μg/g range.
García de Llasera (García de Llasera and Reyes-Reyes 2009)
also reported another MSPD strategy with octadecylsilyl (C18)
sorbent combined with a silica gel cleanup and MeCN elution
was reported in the analysis of chlorpyrifos, chlorfenvinphos,
diazinon, fenitrothion, and parathion-methyl residues in dif-
ferent bovine tissue samples. This method was validated using
LC–DAD determination and GC–MS confirmation, yielding
recovery values higher than 94%, except for chlorfenvinphos in
the liver (55%) with RSDs ≤15% in the liver and ≤11.5% in the
muscle at spiking levels of 0.25, 2.5, and 5 μg/g and LODs below
0.1 μg/g.
OPs were also determined in a multiresidue approach in
olive oil using a combination of liquid partitioning plus MSPD.
Preliminary liquid partitioning with MeCN saturated with petro-
leum ether was followed by MSPD extraction with aminopropyl
and cleanup followed by Florisil cocolumn. Recoveries ranged
from 80% to 120% with RSD <10% with LODs in the 0.003–
0.06 mg/kg range (Ferrer et al. 2005).
Florisil used as the dispersant and EtOAc extraction assisted
by sonication was used in the analysis of nine OPs in fruit juices
using MSPD and GC–NPD determination (Albero et al. 2003).
The average recoveries obtained for all the analyzed pesticides in
the different juices and fortification levels were >70% with RSDs
of <11%. The LODs ranged from 0.1 to 0.6 μg/kg.
Schenck and Wagner (1995) described a rapid technique for
the extraction and GC determination of five OCs and five OP
pesticide residues in milk. Milk (5 mL) was blended with 2.0 g of
C18, and the analytes were extracted with MeCN. Recoveries of
fortified OP pesticide residues (10–50 μg/kg) ranged from 75%
to 105%. The MSPD and the AOAC International multiresidue
Handbook of Food Analysis
method for pesticides in milk produced comparable results for
milk samples containing OC pesticide residues. The use of MSPD
resulted in a 90% reduction in organic solvent consumption and a
95% reduction in the hazardous waste generated when compared
with the AOAC method in the same way, Lehotay et al. (2005)
reported MSPD multiclass method performance when 0.5 g of
sample plus 2 g of C18 and 2 g of anhydrous sodium sulfate were
mixed in a mortar and added above a 2 g Florisil column on a
vacuum manifold. Two portions of 5 mL of MeCN were used to
elute the OP pesticides.
After purification of the extract and fractionation of the two
classes of pesticides with a Florisil solid-phase extraction (SPE)
cartridge, the fraction containing the OP pesticides was analyzed
by GC–FPD, while the fraction containing OC pesticides was
analyzed by GC/ECD.
8.11.1  Microwave-Assisted Extraction
MAE was applied for the first time for the extraction of organic
pollutants in 1986. Recently, it has been successfully used for
the analysis of a wide range of pesticides in many food matrices.
Different from the conventional extraction methods, the analytes
are selectively heated by microwaves and transferred from the
sample to the organic solvents while the food contents are not
extracted (Zhang et  al. 2006). The partitioning of the analytes
from the sample matrix into the extractants depends on the tem-
perature and the nature of the extractant. Unlike classical heating,
microwaves heat the entire sample simultaneously without heating
the vessel; so, the solution reaches its boiling point very quickly,
leading to a very short extraction time (Lambropoulou et al. 2007).
High temperature or microwaves are believed to accelerate
the extraction procedure and enhance the recoveries of the tar-
get compounds. The recoveries increased when the microwave
output power ranged from 20 to 80 W while the recoveries
remained nearly constant within the power range of 80–110 W.
However, excessively high power could result in the degradation
of pesticides and a decrease in recoveries. Extraction times also
influenced the total recoveries, due to possible degradation of the
analytes (Zhang et al. 2006).
The main advantages of MAE are the low required tempera-
ture, high extraction efficiency, complete automation, and the
possibility of simultaneous extraction of different kinds of com-
pounds including these thermal labile compounds. (Ramos 2012;
Zhang et al. 2006). However, the MAE lacks selectivity, result-
ing in the coextraction of interfering compounds and it requires
additional cleanup before the chromatographic determination
(Zhang et al. 2006). Additional drawbacks of this methodology
are the poor efficiency of the microwaves when either the com-
pounds or the solvent are nonpolar or volatile and the difficulty
to integrate MAE devices in a flow system (Lambropoulou et al.
2007; Ramos 2012; Zhang et al. 2006).
A successful MAE method depends on several conditions that
affect the extraction yield, for example, solvent composition, sol-
vent volume, extraction temperature, extraction time, and matrix
characteristics such as water content. The extraction solvents
used usually have high dielectric constant to absorb microwave
energy efficiently; such as MeOH, water, and ethanol. Nonpolar
solvents with a low electric constant, for example, hexane and
toluene, are not potential solvents for this methodology, but their
 Analysis of Organophosphorus Insecticides in Foods
extracting selectivity and efficiency can be modulated by using
mixtures of solvents (Lambropoulou et  al. 2007). The polarity
of the extraction solvent should closely match to that of the ana-
lytes. One of the most commonly used solvents for the MAE of
pesticides in different food matrices are acetone–hexane, ace-
tone, acetone–MeCN (50:50), MeCN–DCM (90:10), and poly-
oxyethylene10 lauryl ether due to the high selectivity and high
recoveries toward the target pesticides (Zhang et al. 2006).
MAE can be conducted with open or closed vessels (focused
microwave-assisted extraction, FMAE, and pressurized micro-
wave-assisted extraction, PMAE, respectively) (Ramos 2012).
In the latter devices, up to 12 extraction vessels can be irradi-
ated simultaneously. PMAE is quite similar to pressurized liq-
uid extraction (PLE), as the solvent is heated and pressurized
in both systems. However, in PMAE, it is necessary to wait for
the temperature to decrease before the vessels can be opened
(Ramos 2012).
Concerning OPs analysis in food, there are few reports related
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to the application of this methodology, maybe due to the need of
performing an additional cleanup after the extraction that makes
the entire procedure tedious. Nevertheless, several authors
report different methods for the analysis of OPs in different food
matrices.
Otake et al. (2012) report the analysis of 16 OP and 10 pyre-
throid pesticides in green onions. The authors tested different
types of solvents for the extraction of the target compounds,
MeCN, hexane, and EtOAc, and two extraction times; 10 and
30 min and 50°C, 70°C, 90°C, and 110°C using a microwave
output power of 1200 W. The best experimental conditions
were MeCN as the extraction solvent, 110°C, and 10 min of
extraction time. The cleanup method used consisted of a liq-
uid–liquid partition of the extract with 10 g NaCl and 0.5 M
phosphate buffer solution (pH 7, 0.20 mL) in a separatory fun-
nel for 10 min. The MeCN layer was dried using anhydrous
sodium sulfate, then it was filtrated, and the remaining sol-
vent was rotary evaporated. The extract was redissolved in
toluene:MeCN (1:3) and it was cleaned up on an SPE ENVI-
Carb/LC–NH2 cartridge that was finally eluted with 10 mL
toluene:MeCN (1:3) followed by concentration to obtain the
final extract. The instrumental analysis was performed by a
GC–MS equipped with a DB-5MS column with the on-column
injection mode and in the selected ion-monitoring mode (SIM).
Under these conditions, the repeatability of the analysis was
satisfactory, in the range from 4.2% to 5.8%. Recovery yields
were in the range of 70%–120% (Otake et al. 2012).
Guillet et  al. (2009) developed a microwave/solid-phase
microextraction (SPME) method for the determination of 23 pes-
ticides including parathion methyl in tomato matrix. Pesticide
extraction from tomato fruits was carried out with a focused
microwave extractor. This method consisted of the extraction
with MeCN: water (50:50) during 5 min at 30 W. Under these
conditions, the sample temperature does not exceed 65°C, to
minimize the possible degradation of thermolabile pesticides.
The extract was centrifuged and a 9 mL aliquot of the superna-
tant was removed for chromatographic analysis. The SPME fiber
was immersed into a stirring solution for 45 min and was then
directly introduced into the chromatographic injector port for
desorption. The chromatographic analyses were performed by
GC–MS with a DB-%MS column in the splitless mode at 250°C
191
in SIM mode. Under these conditions, the reproducibility of the
method was adequate, below 15.7% and the LOD and quantifica-
tion of parathion methyl were 1.21 and 4.05 μg/kg, respectively.
Wang et al. (2007) described a methodology for the analysis of
11 OP pesticides in leeks. This method used microwave heating
to efficiently remove the sulfur interferences and SPE cleanup
prior to GC–FPD analysis. The experimental conditions assayed
were 20 g of homogenized sample leek, heated for 50 s at 650 W.
Then, the sample was extracted with 50 mL of MeCN using an
Ultraturrax. The extract was filtered into a 1000 mL cylinder
containing 5 g NaCl, shaken for 1 min, and allowed to stand for
5 min. A 25 mL aliquot was evaporated under vacuum at 40°C
and dissolved in 3 mL hexane:EtOAc (1:1). Afterward, a cleanup
using a PSA cartridge was performed. The target pesticides were
eluted with 12 mL hexane:EtOAc (1:1). The eluate was collected
and dried with a gentle nitrogen stream for GC–FPD analysis in
the splitless mode using a DB-1701 column. The confirmation of
the pesticides was performed using a GC–MS.
The validation parameters of this method were evaluated at
three concentration levels: 50, 100, and 500 μg/kg. The percent-
ages of recoveries were in the range 73%–111%, whereas the
reproducibility of the studied pesticides ranged from 0.9% to
13.1%. The limits of quantifications (LOQs) were between 2.4
and 12.3 μg/kg except for fenamiphos that presented an LOQ of
30 μg/kg. According to these results, the developed methodol-
ogy presents a good performance for the determination of these
compounds in a difficult matrix such as leek.
In 2008, Fuentes et al. studied the suitability of MAE method
for the determination of OPs in olive oil and avocado oil. The
method is based on microwave-assisted liquid–liquid extrac-
tion with the partition of OP pesticides between MeCN:DCM
(CH2Cl2) (90:10) mixture and oil. A Placket–Burman design,
with two levels for each factor, was developed to assess the effect
of power and time extraction, type and volume of the extracting
solvent, sample amount, and dilution factor with n-hexane. The
final extraction conditions were 5 mL of the extraction solvent
preheated at 250 W for 2 min and then for 8 min at 700 W. The
cleanup of the extracts was performed with ENVI-Carb SPE car-
tridges using CH2Cl2 as the elution solvent. The determination
of the target pesticides was performed with a GC–flame pho-
tometric detection and GC–tandem mass spectrometry, using a
triple-quadrupole mass analyzer, for confirmative purposes. In
2009, the same author developed a method using atmospheric
pressure microwave-assisted liquid–liquid extraction (APMAE)
and SPE or low-temperature precipitation as the cleanup step for
the determination of the same pesticides.
Wang et  al. (2007) described a method of MAE followed by
GC for the determination of OP pesticides in rice. The optimum
conditions for extraction solvents and MAE time were studied.
The linear ranges of the method were from 10 to 800 μg/kg. The
detection limits were from 1 to 5 μg/kg. The recoveries were from
73.6% to 94.5%, and the RSDs were from 1.5% to 7.5% (n = 3).
This method has proved to be rapid, efficient, and accurate.
Yang et  al. (2002) developed an MAE method followed by
GC–MS for the determination of three OPs in vegetables. Several
extraction solvents were compared on the MAE efficiency.
CH2Cl2 gave good recovery, and was chosen for the procedure.
A three-level orthogonal array design was used to optimize
the MAE process. Factors affecting the MAE efficiency were
 192
considered, including the solvent amount and extraction time.
The linear ranges of the method were from 4 to 400 μg/kg for
diazinon and parathion, and from 20 to 400 μg/kg for isocar-
bophos. The detection limits were below 5 μg/kg. Two sets of
spiked vegetable samples of 200 and 50 μg/kg were determined.
The recoveries were from 72.2% to 102.0%, and the RSDs
were from 1.5% to 11.0% (n = 3). The analytical results agreed
quite well with those obtained by conventional extraction with
mechanical shaking (EMS).
8.11.1.1  Solid-Phase Microextraction
SPME is a sample preparation technique developed by Arthur and
Pawliszyn in 1989. Since then, it has been widely applied in dif-
ferent matrices such as environmental, food, and biological, com-
bining both GC and high-performance liquid chromatography
(HPLC). It is a convenient alternative to other extraction meth-
ods because it integrates sampling, extraction, concentration, and
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015
sample introduction into a single step without the use of a solvent.
SPME is mainly based on the use of a fused-silica fiber, coated
with a polymeric stationary phase and mounted on a modified
GC syringe. There are two ways of working with SPME. In
direct immersion SPME (DI-SPME), the extraction of the target
analytes from the sample to the fiber is realized with the coated
fiber immersed in the liquid sample, while in headspace SPME
(HS-SPME), the fiber is exposed to the atmosphere of the vial,
above the sample.
Several factors infuence the extraction of analytes; some of
these are directly related to the SPME device such as coating
volume and characteristics. Sample parameters must also be
kept constant; these include sample volume and vial size, extrac-
tion temperature, agitation, and extraction time. Finally, all the
adsorption and desorption factors must be consistent including
the period and depth of immersion, time between immersion and
exposure to the GC, and GC desorption parameters including
temperature and time.
Regarding the analysis of OPPs in food matrices, there are
many studies that probe that SPME is an excellent alternative
and it has been successfully applied in diverse situations.
In 2013, Zi-Ye et al. developed an HS-SPME–GC–MS method
for the assessment of 11 OPPs in multiple vegetable and fruit
commodities. In this work, the authors studied seven characteris-
tic commodities (turnip, green cabbage, French beans, eggplant,
apple, nectarine, and grapes) cataloged in Codex Alimentarius
Commission. The 11 OPPs were phorate, diazinon, disulfoton,
chlorpyrifos methyl, fenchlorphos, pirimiphos methyl, chlorpy-
rifos, isofenphos, phenthoate, prothiophos, and ethion. The sam-
ple treatment consisted of the addition of 100 μL of methanol/
acetone (1:1 v/v) extraction solvent and 10% (w/v) aqueous NaCl
solution to 1.0 g of homogenized sample in a 20 mL glass vial.
The vial was then warmed at 70°C in a water bath for 10 min.
Finally, the fiber was exposed to the headspace for 45 min at
the same temperature and after sampling; it was inserted into
the GC injection port (240°C) for 7 min in the splitless mode.
SPME experiments were performed using a polydimethylsilox-
ane (PDMS, 100 μm) fiber and the concentration range evalu-
ated was from 0.02 to 2.5 mg/L. The method validation was
carried out by the determination of linearity, sensitivity (LOD
and LOQ), precision, and accuracy.
Handbook of Food Analysis
Lingshuang et al. (2006) studied the use of a vinyl crown ether
fiber for the determination of eight OPPs in food samples (apple
juice, apple, and tomato) with GC coupled to flame photomet-
ric detector (GC-FPD). Five kinds of fibers were analyzed (three
kinds of vinyl crown ether polar fibers prepared with sol–gel pro-
cess, and the commercial 85 μm polyacrylate [PA] and 65 μm
PDMS–divinylbenzene [DVB]) with the home-made ones show-
ing the best result in terms of extraction efficiency and sensi-
tivity for the studied analytes. DI-SPME against HS-SPME was
also evaluated, obtaining a higher extracted amount of almost all
OPPs in HS-SPME. Sample treatment of apple juice consisted
of the dilution with pure water (1:30 w/v) and addition of 5 g
of NaCl. An aliquot of 15 mL was placed in a 25 mL vial and
stirred with a magnetic bar at 70°C. The same sample treatment
was done for apple and tomato homogenate, with variation in the
dilution, being 1:50 and 1:70, respectively. The fiber was exposed
to the headspace during 45 min and then inserted into the GC
injection port during 5 min (desorption time) at 270°C. The
method linearity was evaluated between 0.01 and 100 mg/kg.
Recoveries ranged from 71.5% to 104.6% for apple juice; 70.5%
to 104.9% for apple, and between 55.3% and 105.3% for tomato
at the three levels assayed (5, 10, and 20 μg/kg) with RSD values
below 11.2%.
Yi-Long et al. (2008) reported the use of a home-made fiber (co-
poly [hydroxyl-terminated silicone divinylbenzene]; OH-TSO/
DVB, 65 μm) for the determination of five OPPs (dichlorvos,
diazinon, methyl parathion, malathion, and ethyl parathion) in
pakchoi samples by HS-SPME coupled with gas chromatogra-
phy with nitrogen–phosphorus detector (GC-NPD). The authors
optimized the following parameters: extraction temperature,
extraction time, ionic strength, constant agitation, effect of the
sample matrix, desorption temperature, and desorption time.
Prior to SPME analysis, 20 g of pakchoi was cut and homoge-
nized with 60 mL of double-distilled water and this homogenate
was finally diluted with water (1:20 w/w). For the SPME process,
5 mL of vegetable sample and a stirring bar were placed in a
presilanized vial. The optimum parameters selected for analysis
were: extraction temperature, 75°C; extraction time, 50 min; no
salt addition; constant agitation; desorption temperature, 270°C;
and desorption time, 3 min. Recoveries of the studied analytes
were in the range of 80.7–101.5%. LODs were between 0.007
(parathion ethyl) and 0.07 μg/kg (dichlorvos).
A HS-SPME–GC–MS methodology was developed by
Dimitra and Triantafyllos(2003) for the rapid screening of seven
OPP (diazinon, fenitrothion, fenthion, parathion ethyl, bromo-
phos ethyl, bromophos methyl, and ethion) residues in strawber-
ries and cherries. The sample preparation involved the use of
5 g of the strawberry and cherry sample containing 15% (w/v)
Na2SO4 and an adequate amount of water (water/pulp ratio 1/2
for strawberries and 1/3 for cherries) in a 20 and 25 mL vial,
respectively. After equilibration at 75°C for 10 min, the fiber was
exposed to the headspace above the sample for 45 min with mag-
netic stirring. Thermal desorption of the analytes was achieved
by inserting the sorbent fiber (PDMS, 100 μm) into the injection
port (held at 240°C) for 5 min. GC analysis was carried out using
a Shimadzu model QP 5000 GC–MS in SIM mode. The authors
described the need of adding water and solvent (methanol) to
the samples to favor the release of the analyte from the matrix.
The ratio of solvent/water at 2.5% (v/v) was selected. Linearity
 Analysis of Organophosphorus Insecticides in Foods
experiments were carried out with fruit samples in a concentra-
tion range from 50 to 500 μg/ kg showing regression coefficients
between 0.986 and 0.994. Three spiked levels were assayed (75,
150, and 300 μg/kg) and recoveries were in the range of 76%–
94% for strawberries and 74%–90% for cherries. In both matri-
ces, RSD values were below 15%. LODs were below 13 μg/kg.
This methodology was compared with liquid–liquid extraction
obtaining similar results. Therefore, HS-SPME procedure can be
proposed as a fast and accurate methodology for analyzing OPPs
in these matrices.
Tsoutsi et  al. (2006) developed a screening method for the
analysis of nine OPPs (dimethoate, diazinon, fenitrothion, mala-
thion, fenthion, parathion ethyl, methyl bromophos, methida-
thion, and ethion) and four metabolites (omethoate, malaoxon,
SO, and fenthion sulfone) in olive oil samples based on HS-SPME
coupled with GC with flame thermoionic detector (GC-FTD).
They compared the efficiency of six commercial fibers (75 μm
CAR/PDMS; 85 μm CAR/PDMS; 7 μm PDMS; 30 μm PDMS;
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015
100 μm PDMS; and 65 μm PDMS–DVB) resulting in the 100 μm
PDMS that was the most suitable for the analysis due to the fact
that it had better extraction efficiency for the majority of target
analytes. Approximately, 5 g of olive oil was placed into 10 mL
crimptop headspace vials, capped with polytetrafluoroethylene
(PTFE)–gray butyl-coated septa. The samples were heated at
75 ± 1°C with magnetic stirring (0.8-cm-long PTFE-coated stir
bar, 960 rpm) during 10 min (equilibration time). After this time,
the needle of the SPME device pierced the septum of the vial
and the fiber was immersed into the headspace of the sample for
60 min, 1 cm above the sample. The thermal desorption of the
analytes was achieved by inserting the fiber into the injection
port (held at 250°C) for 7 min. Calibration curves were drawn
for five levels of concentration in the range 0.025–0.50 mg/kg
with correlation coefficients higher than 0.9856. The matrix
effect analyzing oil samples with different composition (acidity,
fatty acids, triglycerides, and sterols) was also evaluated. They
found that only acidity and the total amount of sterols were the
main factors influencing SPME efficiency concluding that each
olive oil sample had to be considered a unique matrix. LODs
ranged between 0.006 and 0.010 mg/kg with LOQs from 0.016 to
0.030 mg/kg. The recoveries of the extraction procedure at three
fortification levels (0.080, 0.16, and 0.32 mg/kg) ranged between
80% and 106% for all the analytes studied with RSD values
below 12%. Repeatability (intra-day precision), expressed as
RSD (%) values, was below 5% while reproducibility (inter-day
precision) never exceeded 10%. The performance was tested in
30 olive oil samples, from the island of Crete in Greece. Fenthion
and SO (from 0.055 to 0.13 mg/kg), ethion (0.011–0.093 mg/kg),
dimethoate, and omethoate (0.022–0.044 mg/kg) residues were
found.
González-Rodríguez et  al. (2005) evaluated the determina-
tion of 14 OP residues, among other families, in different kinds
of milk combining SPME and low-pressure gas chromatogra-
phy–tandem mass spectrometry (LP-GC–MS/MS). The authors
worked with different kinds of processed (whole, skimmed,
and powdered) and unprocessed (goat and human) milk sam-
ples. Sample pretreatment consisted of mixing 1 mL aliquots
of milk with 500 μL of formic acid, 10 μL of triethylenamine
(TEA), and 50 μL of 2 mg/L pentachlorobenzene in acetone
solution. The mixture was centrifuged for 10 min at 3000 rpm.
193
Approximately, 1 mL of the superior layer was transferred to a
test tube, where 2490 μL of milli-Q water was added and mixed.
The sample was then centrifuged again for 2 min (3000 rpm)
and 1 mL of the superior layer was transferred to a GC vial for
SPME extraction. HS and DI- SPME were evaluated for analyz-
ing the target pesticides. For HS-SPME, two coating fibers were
evaluated: 100 μm PDMS and 65 μm PDMS–DVB. The heating
temperature was set to 70°C. The adsorption time was 40 and
45 min for PDMS and PDMS–DVB, respectively. Desorption
time was varied from 5 to 20 min and finally set to 15 min for
both types of fibers. Better results were obtained adding 0.1 g of
NaCl. DI-SPME analysis was also optimized using the same way
as done for HS-SPME. After some preliminary experiments, DI
of the fiber was discarded due to the fact that results had poor
repeatability and sensitivity. The authors considered the dilution
of milk samples at various levels (1:2, 1:3, 1:4, and 1:5 v/v) finally
selecting a 1:4 v/v dilution. After optimization, desorption time
was set at 40 and 55 min for PDMS and PDMS–DVB fibers with
the same conditions for HS-SPME. Best results were obtained
without adding salt. PDMS–DVB fiber was selected for this
experiment in terms of sensitivity. Comparing between HS and
DI-SPME, a better sensitivity was achieved by immersing the
fiber in the diluted milk than by using HS-SPME. At the tested
concentration, seven OPs (parathion methyl, malathion, fenthion,
parathion ethyl, chlorfenvinphos, quinalphos, and fenamiphos)
were not detected. The DI-SPME–LC-GC–MS/MS method was
validated studying LOD and LOQ, linearity, trueness and pre-
cision, and uncertainty. LOD values ranged between 0.01 and
0.19 μg/L while LOQ ranged from 0.02 to 0.65 μg/L. Linearity
was studied in the range 1–50 μg/ L using three calibration lev-
els and the origin (calibration curve was not forced through the
origin). Recovery data were obtained by analyzing aliquots of an
uncontaminated milk (n = 10) spiked at 20 μg/L. Mean recovery
rates were in the range 87.1%–103.2% with RSD values below
11.5%. The uncertainty was never >25.5% at the lowest calibra-
tion point for all concentration levels. This methodology was
applied to the analysis of real milk samples: commercial (pow-
der and pasteurized), and goat and breast human samples. OP
residues were not found in any samples.
Fernández et  al. (2001) established an SPME–GC–NPD
method for the simultaneous determination of 18 OPs residues
in honeybee samples (Apis mellifera). Two SPME fibers were
compared: 85 μm PA phase and 100 μm PDMS phase. The
honeybee samples were first lyophilized to eliminate the matrix
putrefaction process and derive a better extraction solution.
Three grams of sample (~10 g of honeybees, fresh weight) were
pounded with a glass pestle in a sifter to obtain a homogeneous
sample. Samples were spiked with 100 μL of a solution contain-
ing 10 mg/L of each pesticide and were allowed to stand at room
temperature for 2 h. The sample was then diluted with 50 mL
of a solution containing acetone–water (1:1 v/v). Afterward, the
solution was shaken vigorously for 30 min and filtered through a
10 g Celite layer on a Butchner funnel with filter paper. 300 μL
of the filtrate was diluted in 3 mL of water solution and placed in
a 4 mL sample vial. Solutions were stirred at 700 rpm. Different
parameters (immersion time, extraction temperature, and ionic
strength) were optimized. PA fiber showed to extract a greater
amount of target analytes than PDMS fiber, because of the stron-
ger affinity of polar analytes such as OP compounds for PA fiber.
 194
Therefore, optimal extraction was achieved with 85 μm PA. An
immersion time of 45 min was selected. The temperature effect
was evaluated by varying the temperature from 25°C to 60°C.
An increase in extraction temperature caused an increase in the
extraction rate and a simultaneous decrease in the distribution
constant between the analytes and the fiber. The effects of this
parameter varied considerably among the different pesticides;
therefore, the analysis was performed without considering the
effect of temperature. The effect of ionic strength on extraction
efficiency was also evaluated but due to the wide range of hydro-
phobicities and opposite behaviors, no salt modifications were
considered. Recoveries were assayed at four levels between 0.01
and 0.2 mg/kg obtaining values in the range from 73% to 128%
for most of the analytes. Bromophos and pirimiphos methyl pre-
sented low recoveries while methidation showed a percentage
above 200% at three levels. Regression coefficients ranged from
0.9616 to 0.9996. Precision was between 1% and 13% except
for methidation (30%) due to matrix interferences. LOD was
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015
between 1 and 10 μg/kg.
8.12 Instrumental Determination of
Organophosphate Pesticide Residues in Foods
8.12.1  Thin-Layer Chromatography
Thin-layer chromatography (TLC) is the oldest but also the
cheapest of all OP analytical methods. This technique is par-
ticularly useful in the case of acetyl cholinesterase inhibitors as
the enzyme associated to a fluorescent reactive is a very sensi-
tive method to determine insecticides such as carbamates and
OPs. Routinely, silica plates are spotted with 50 mL of a solu-
tion of the sample suitably prepared and developed with ethyl
acetate or mixtures of hexane: ethyl ether or DCM. The plate
is then sprayed with a solution of acetylcholinesterase, incu-
bated at 37°C, and then sprayed with a suitable color-develop-
ing agent, an ester that can be differentially visualized when
it is intact or it has been hydrolyzed by the enzyme. The most
common reagents are β-naphtholacetate or acetylthiocholine.
The method can be performed quantitatively coupled to a suit-
able densitometer, reaching LODs of 0.005 mg/kg and LOQs of
0.02 mg/kg but its main application is for screening purposes
(Ambrus et al. 2005).
8.13  Gas Chromatography
The instrumental determination of OPs has been dominated by
the GC separation of the pesticide mixture, and the compounds
detected with specific, selective, or universal detectors.
According to the levels that have to be determined, only a few
GC detectors have proven to be useful for OP determination.
8.13.1  Conventional Detectors for OPs
8.13.1.1  Flame Photometric Detector
The FPD is a specific detector for phosphorus- and sulfur-contain-
ing compounds. The analytes eluting from the chromatographic
Handbook of Food Analysis
column are burnt within a hydrogen flame and the luminescence
of elementary phosphorus is detected and measured through
a λ = 526 nm filter in a photomultiplicator. This detector is a
sensitive one that allows the determination of ­femtograms in
the detector flame for phosphorus-containing compounds. The
relationship luminescence versus concentration is linear for
phosphorus, whereas at λ = 394 nm, the relationship lumines-
cence toward concentration is quadratic for sulfur-­containing
compounds. This detector has a sensibility 100.000 times higher
toward sulfur and/or phosphorus than luminescence due to car-
bon at these wavelengths, providing enough selectivity to detect
compounds with phosphorus and sulfur in the presence of other
coeluting compounds from the matrix. The sensitivity of the
detector is high permitting levels of detection and quantification
up to 0.005 and 0.02 mg/kg, respectively when coupled to suit-
able sample preparation procedures (Patel et al. 2004; Martínez
Salvador et al. 2006).
The main feature of the FPD detector is that it allows a high
sample concentration factor due to the high specificity of the
detection method that minimizes the matrix signals. Even the
popular QuEChERS sample preparation procedure has a version
for conventional detectors.
8.13.1.2  Nitrogen–Phosphorus Detector
NPD is a popular detector as it allows the simultaneous determina-
tion of phosphorus- and nitrogen-containing compounds. It is an
FPD detector where an alkaline pearl of CsBr is burnt, stabiliz-
ing a current specific for P- and N- containing ions, with great
selectivity and sensitivity and also with remarkable robustness.
It has been widely used in pesticide residue analysis as most of
the GC-amenable compounds contain either nitrogen or phos-
phorus atoms. There are hundreds of reports in the literature that
employed this detector to the quantification of minute amounts of
pesticide residues in food commodities. A representative study of
the usefulness of this detector for the determination of organo-
phosphorus insecticides is its application in the work reported by
Uygun, where the dissipation of eight OPs in different wheat meals
during pasta processing was studied (Uygun et al. 2008).
8.13.1.3  Electron Capture Detector
The ECD is a very sensitive one that allows the determination of
fg amounts of chemicals in different samples. Compounds eluting
from the GC column pass through a zone where a beam of b is
beta particles is continuously emitted. Electron-active compounds
cause changes in the electron flux that are recorded as a perturba-
tion of the baseline current. Only OPs bearing either sulfur or aro-
matic residues or halogen atoms can be detected. Therefore, some
common OPs can be easily detected, mostly thiophosphates, (eth-
ion, parathion, and malathion), and halogenated pesticides (chlor-
pyrifos) but it has little sensitivity toward diazinon.
8.14 Mass Spectrometric Determination of
Pesticide Residues
All the above-mentioned detectors need an extra confirmation
method to ascertain the identification of the detected residue.
 Analysis of Organophosphorus Insecticides in Foods 195

For years, multiple column separations, and crossing of detec- Table 8.4
tion methods was required to unequivocally confirm the find- Quantifier and Qualifier Ions of OP Insecticides
ings in the different samples analyzed. The development of mass in GC/MS
spectrometer detectors allowed the qualitative identification and
Quantitation Qualifier
therefore the confirmation of the pesticide detected as well as the
Pesticide Ion (m/z) Ions (m/z)s
precise quantification of the residues. Nowadays, the mass spec-
trometric study is the method of choice for the determination of Omethoathe 156 126,110
the residue as the identification and quantification steps are per- Dimethoathe 93 125,143
formed simultaneously. Three independent criteria are met that Methamidophos 94 141,64
speed out the analysis. The retention time, the MW, and the frag- Acephate 136 94,125
mentation of the molecules are unique properties of each com- Fenthion 278 125,109
pound that are combined to reach the objective (SANCO 2013). Malathion 173 127,93
Azinphos methyl 160 125,132
Diazinon 179 304,152
8.14.1 Gas Chromatography–Mass Spectrometry Chlorpyrifos 258 127,314
GC was the first separation method that could be coupled to mass Parathion methyl 291 125,97
spectrometers. Compounds eluting from the chromatographic col- Ethion 231 384,153
umn are already in the vapor phase allowing their direct entrance Pirimiphos methyl 180 266,310
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015

to the ionization chamber, where the compound is fragmented,

usually after colliding with a high-energy (70 eV) electron beam
(EI mode). The spectra thus produced are the same that have been
recorded for years in magnetic sector spectrometers and can be
compared with those already existing in commercial libraries
such as the one from National Institute of Technology (NIST).
Expensive sector spectrometers were replaced by cheaper and
more versatile quadrupole spectrometer detectors. Benchtop GC
or LC/MS were built and the applications of mass spectrometry
to the detection of trace compounds boosted up.
Pesticide residue analysis through mass spectrometry requires
the identification and confirmation of the detected compound, as
stated above. Full EI spectrum is the fingerprint of a compound and
the fitting of an unknown with one already recorded in a library,
allows its identification. However, for trace compounds, many
times, only a portion of the spectra could be recorded, making
identification more complex. Therefore, residue analysis of trace
compounds is rather limited using full spectrum identification, as
well as in the quantification step. The system scans for the whole
range of masses, the sensitivity is lost, and detection limits rise up.
The problem has been solved fixing the lens to a specific mass that
is monitored continuously. This recording mode is called SIM, sin-
gle-ion-monitoring, mode that also permits a direct quantification
of the compound. The mass spectrometer can record many ions at
the same time and other characteristic ions of the molecule can be
simultaneously registered. The relationship between the intensities
of the recorded ions is another constant for a given compound, thus
providing an extra criterion for compound identification. The ion
used for quantification is called the “quantifier” and those used for
identification are the “qualifier” ones.
In simple GC–MS, these criteria have been widely applied to
the determination of organophosphate residues. Characteristic
ions of important OPs are summarized in Table 8.4.
Soft ionization techniques (chemical ionization, CI, atmo-
spheric chemical ionization, APCI, and electron spray ioniza-
tion, ESI, among others) provide easy access to molecular ions,
which cannot be achieved many times through EI at 70 eV. These
quasi-molecular ions can be further fragmented to yield charac-
teristic fragment ions. Therefore, tandem MS/MS experiments
can be performed, and, selecting the ions properly, selectivity
and specificity are greatly increased (see Figure 8.9).
The ion traps (ITD) were the first configurations of mass
spectrometers that permitted MS n experiments to be done.
Nevertheless, the great drawback of these MS detectors is that
there is no library available but those that each laboratory built
itself. Although the detection limits were lowered and the level
of microgram per kilogram for pesticide residues was easily
reached, GC–ITD equipment did not replace the completely
simple GC–MS configuration in pesticide residue analysis. In
the first decade of the century, most of the European laborato-
ries that participated in the proficiency tests organized by the
EU Reference Laboratory were equipped with GC hyphenated
to conventional detectors and simple GC–MS. Only with the
advent of triple quadrupoles, GC determination of pesticide
residues moved definitively to tandem MS. Triple quadrupoles
are the most robust configuration to perform such experiments,
working in the multi-reaction-monitoring mode. The ioniza-
tion technique EI (70 eV), ESI, CI, or low-energy EI (20 eV)
renders an ion that is selected in Q1 that yields, after collision
with gas molecules in Q2 fragment ions from the selected par-
ent ion. These are focused and recorded in the third quadru-
pole Q3. The parent and fragment ions are specific for a given
molecule as well as their relative intensities. The sum of all
these parameters allows the identification and quantitation of
a certain trace compound in a mixture. Table 8.5 shows the
criteria for the identification of a compound through MS sug-
gested by the SANCO document No/12571/2013.
The third criteria, the acceptable range of relative intensities
between parent and fragment ions as well as between the ions
selected for monitoring has also been ruled out by the SANCO
guidelines.
8.15  Liquid Chromatography/Mass Spectrometry
HPLC coupled to conventional detectors, has never been com-
petitive with OPs GC determination.
The coupling between liquid chromatography and mass spec-
trometry has been successfully performed some 20 years after the
development of GC/MS. Despite the fact that its development is
 196 Handbook of Food Analysis

100 1
90 RT: 9.48
80 AA: 44388

Relative abundance
70 SN: 8
60
50
40
30
20
10
0
9.3 9.4 9.5 9.6 9.7 9.8 9.9 10.0 10.1 10.2 10.3
Time (min)

100 67.4
2
79.4
80
Relative abundance

91.4
60
313.5
135.2
40 97.3 315.5
199.1
147.2171.1 213.0 258.1285.6 354.6
428.3
20 355.6 413.5
316.4 430.3462.7 502.0
0
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015

50 100 150 200 250 300 350 400 450 500 550 600 m/z

100
RT: 9.48 3
80 AA: 11034
Relative abundance

SN: 71
60

40

20

0
6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5
Time (min)

258 4
100
Relative abundance

80

60 259
40
286
20

0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 m/z

Figure 8.9  Comparison between different MS modes of chlorpyrifos determination in pepper. (1) GC/MS full scan trace of the pepper extract. (2) Mass
spectrum of the peak at tr = 9.48 min, suspected, and chlorpyrifos. (3) GC–MS/MS (ITD) trace of the same extract at tr = 9.48 min. (4) Selected ions for
chlorpyrifos identification and quantitation (see Table 8.5). (From A.R. Fernandez Alba, personal communication.)

much recent, the improvements made in instrumentation and detec- for identification and quantification are retention time and the
tion systems, turn the LC/MS/MS one of the most popular methods compound exact mass, which is a unique molecular feature of
for pesticide residue analysis as all the nonvolatile compounds that each compound (Ferrer et  al. 2005). An automatic database
could not be studied by GC could be determined through LC. search grounded on the easily calculated pesticide exact masses
The ionization modes in liquid chromatography/mass spec- had been proposed (Thurman et al. 2005). Table 8.6 shows the
trometry (LC/MS) are soft ones, and pesticide extracts are more molecular formula and exact masses for some OPs.
complex mixtures than those observed during GC analysis. For The criteria for the mass determination is that the devia-
this reason, simple LC/MS has limited application in pesticide res- tion from the measured masses should not exceed 5 ppm;
idue analysis. Quasi-molecular ions are generated for most com- meaning that the narrower the width of the mass peak, the
pounds, and further fragmentation can be achieved increasing the more exact the measured mass. The main drawback for TOF
fragmentor voltage. The drawback is that matrix coextractives are measurements is that the sensibility of the TOF equipment is
also ionized and detected, hampering the pesticide determination. enough for screening purposes but only the new instruments
LC/MS time of flight (TOF) is also a suitable method for pes- are capable of performing quantitative determination at the
ticide residue analysis. In this case, the two criteria employed lowest MRLs.
 Analysis of Organophosphorus Insecticides in Foods 197

Table 8.5 Table 8.7

Criteria for Compound Identification through Mass Spectrometry Sensitivity of GC–(EI) MS and LC(ESI)MS toward OPs
Detection
Pesticide GC (EI) LC (ESI+)
Single MS Single MS High
(Standard Mass Mass Acephate + +++
MS Mode Resolution) Resolution MS/MS Azinphos methyl + ++++
Bromophos methyl +++ +++
Systems Quadrupole, ion trap, TOF, Orbitrap, QqQ, qtrap, and
and TOF FTMS, and QTOF Chlorfenvinphos +++ +++
magnetic sector Chlorpirifos +++ ++++
Acquisition Full scan, limited Full scan, Selected/ Chlorpirifos methyl +++ +++
mass range, and limited mass multiple-reaction Coumaphos +++ ++
SIM range, and SIM monitoring Diazinon ++++ ++++
Requirements ≥3 dignostic ions ≥2 diagnostic ≥2 product ions Dimethoate +++ ++++
for (preferably ions Ethion +++ ++++
identifi­ including
cation quasi-molecular ion)
Fenchlorphos +++ ++++
Fenitrothion +++ ++
Source: SANCO document No/12571/2013.
Fenthion ++ ++
Downloaded by [Horacio Heinzen] at 06:35 19 September 2015

Table 8.6 Malaoxon +++ +++

Malathion +++ +++
Molecular Formula and Exact Masses for Some OPs
Metamidophos + ++++
Pesticide Molecular Formula Exact Mass Methidathion ++ ++++
Dimethoate C5H12NO3PS2 230.0069 Omethoate + ++++
Diazinon C12H21N2O3PS 305.10832 Parathion ethyl +++ +++
Malathion C10H19O6PS2 331.04334 Parathion methyl +++ ++
Methidathion C6H11N2O4PS3 324.95108
Parathion ethyl +++ ++
Phenamiphos + ++++
Pirimiphos methyl +++ +
Propetamphos +++ ++++
8.16  Liquid Chromatography Coupled to MS/MS Pyriproxyfen +++ ++++
LC-MS/MS equipment allows the selective detection and quanti- Tetradifon ++ ++++
fication of trace amounts of pesticides. The process of MS/MS in
the MRM mode yields different ions than those observed in sim-
ple EI (70 eV) ionization. The mass of the fragments observed moved toward LC. Some OPs do not have good sensibility in LC,
depends on the energy of the collision gas in Q2. particularly those bearing a very electronegative Y group (Figure
8.1) such as parathion methyl or pirimiphos ethyl. On the other
hand, polar compounds such as metamidophos, acephate, and
Pesticides Precursor Ion Fragment Ion
azinphos methyl are preferably analyzed by LC(ESI) MS/MS.
Omethoathe [M+H]+ 214 109, 125, 183 Table 8.7 shows a qualitative classification of the sensitivity of
Dimethoate [M+H]+ 230 125, 199, 171 LC and GC toward OP compounds.
Methamidophos [M+H]+ 142 125, 94, 112
Acephate [M+H]+ 184 125, 143, 113
Fenthion [M+H]+ 279 169, 247, 205
Malathion [M+H]+ 331 127, 99, 285 8.17 Conclusions
Azinphos methyl [M+H]+ 318 132, 160, 125
OPs are still a very important class of pesticides, widely employed
Diazinon [M+H]+ 305 169, 97, 153
for crop and animal protection. Their toxicity slowly forces regu-
Chlorpyrifos [M+H]+ 352 200, 97, 198
latory authorities to ban some pesticides all around the world
Parathion methyl [M+H]+ 264 125, 232, 79
Ethion 385/402 199/385,199
such as diazinon in the United States (Shah and Iqbal 2010).
[M+H]+/[M+NH4]+
Pirimiphos methyl [M+H]+ 306 108, 164, 67 Nevertheless, laboratories of pesticide residues seeking food
safety must have these compounds within their scope as there
are huge reserves of these compounds and, as it happened with
The boost in pesticide residue analysis has been triggered by the the OC pesticides of which there are still tons stored, it could be
possibility of performing MS/MS experiments, as already men- foreseen that their use will continue for years, banned or not. The
tioned. Methods describing the simultaneous determination of up new concepts of sample miniaturization and the aim to deter-
to 600 pesticides belonging to all families and classes have been mine a wide scope of analytes in a single run (using very sensi-
described. OPs are GC-amenable compounds as described pre- tive mass-spectrometry based methods) has drifted the interest
viously that can also be analyzed through LC. Inspection on the from pesticide residue-class analysis to multiresidue methods.
reports of the European proficiency tests of pesticide residues on In these multiresidue protocols, OP residues determination is of
fruits and vegetables showed that OP analysis has not completely paramount importance to protect public health.
 198 Handbook of Food Analysis

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