LC-MS/MS Determination of Pesticide

Residues in Fruits and Vegetables 72

Anna Stachniuk

Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2139
2 Pesticides: General Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2140
3 Pesticide Legislation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2143
4 Approach to Pesticide Identification by Means of LC-MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . 2143
4.1 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2144
4.2 Sample Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2147
5 The Most Frequently Detected Pesticides in Fruits and Vegetables . . . . . . . . . . . . . . . . . . . . . . . 2150
6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2155
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2156

Abstract
A proper diet is commonly regarded as one of the most important factors
determining one’s health. A key role in such a diet is played by unprocessed
food of plant origin, mainly fruits and vegetables, as they contain many important
dietary bioactive compounds such as polyphenols, carotenoids, fiber, antioxi-
dants, several important vitamins, and minerals. Despite the nutritional benefit,
they may also contain substances that adversely affect human health. Pesticides
constitute a special group of contaminants as even small amounts of these sub-
stances can result in acute poisoning, lead to cancer, and have an adverse impact
on the endocrine, immune, and nervous system. A considerable number of
pesticides have a harmful effect already in low concentrations, within the range
of μg kg 1 and below μg kg 1; hence, there is a great need for identifying and
determining them by means of highly selective and sensitive methods. In the
analysis of pesticide residue, similarly to the analysis of other food contaminants,

A. Stachniuk (*)
Laboratory of Separation and Spectroscopic Method Applications, Center for Interdisciplinary
Research, The John Paul II Catholic University of Lublin, Lublin, Poland
e-mail: [email protected]

# Springer Nature Switzerland AG 2019 2137

J.-M. Mérillon, K. G. Ramawat (eds.), Bioactive Molecules in Food, Reference Series in
Phytochemistry, https://doi.org/10.1007/978-3-319-78030-6_82
2138 A. Stachniuk

there is a clear tendency to prepare multiresidue methods that enable monitoring a

large number of compounds in a great number and variety of samples. Most
multiresidue methods reported for fruits and vegetables in the last decade are
based mostly on the use of liquid chromatography coupled with tandem mass
spectrometry (LC-MS/MS), which is the technique of choice for the majority of
pesticides and their metabolites nowadays.

Keywords
Pesticides · Fruits and Vegetables · Liquid chromatography–mass spectrometry

Abbreviations
APCI Atmospheric pressure chemical ionization
API Atmospheric pressure ionization
ASE Accelerated solvent extraction
C18 Octadecylated silica bounded stationary phase
C8 Octasilyl silica bounded stationary phase
CHEMAC Conservative homogenizing extraction and multifunction adsorp-
tion cleanup
CID Collision-induced dissociation
CNTs Carbon nanotubes
dSPE Dispersive solid phase extraction
ESI Electrospray ionization
EU European union
GC Gas chromatography
GCB Graphitized carbon black
GC-MS Gas chromatography coupled with mass spectrometer
GC-MS/MS Gas chromatography coupled with tandem mass spectrometer
HPLC High-performance liquid chromatography
HRMS High-resolution mass spectrometry
LC-MS Liquid chromatography coupled with mass spectrometer
LC-MS/MS Liquid chromatography coupled with tandem mass spectrometer
LLE Liquid–liquid extraction
MAE Microwave-assisted extraction
MRL Maximum residue level
MRM Multiple reaction monitoring
MS Mass spectrometry
MSPD Matrix solid phase dispersion
MWCNTs Multiwalled carbon nanotubes
OPPs Organophospohorus pesticides
PSA Primary–secondary amine
Q Quadrupole
QQQ Triple quadrupoles
Q-TOF Quadrupole-time of flight
Q-Trap Quadrupole-linear ion trap
72 LC-MS/MS Determination of Pesticide Residues in Fruits and Vegetables 2139

QuEChERS Quick, easy, cheap, effective, rugged, safe

SCAN Scan monitoring
SFE Supercritical-fluid extraction
SIM Selected ion monitoring
SPE Solid phase extraction
SPME Solid phase microextraction
SRM Selected reaction monitoring
STEMIT Single-tube extraction with multisorbent impurity trapping
SWCNTs Single-walled carbon nanotubes
TIC Total ion current
TOF Time of flight
UHPLC Ultrahigh-performance liquid chromatography
WHO World health organization

1 Introduction

Recent years have seen a clear trend encouraging people to consume unprocessed
food. An active lifestyle and a balanced diet have a decisive impact on protecting
human health. Fresh fruits and vegetables hold a prominent place in this diet as they
are a rich source of fiber and several vitamins and minerals important for health.
According to the World Health Organization (WHO), consumption of fruits and
vegetables in Europe constituted over 30% of consumer diet [1]. In unprocessed
food, including raw fruits and vegetables, a considerable number of healthy bioactive
compounds can be accompanied by contaminants occurring in atmospheric air, soil,
and water. Unfortunately, almost all of the agricultural commodities currently culti-
vated and distributed are exposed to contaminants. Three main groups of contami-
nants are distinguished based on the impact of environmental factors: physical,
chemical, and biological [2]. Chemical contamination, mainly caused by the increas-
ing use of agrochemicals in the agricultural and food industry, constitutes a special
group of contaminants. Unlike dangerous biological contamination of food immedi-
ately resulting in poisoning, chemical contaminants usually emerge with a delay
because they can accumulate in human tissue over a long time and cause symptoms
of disease only after reaching a critical level. The presence of chemical contaminants
in food is one of the basic criteria for assessing the safety and quality of foodstuffs [2].
Plant protection products, i.e., pesticides, constitute the most varied source of food
contaminants. Owing to their long-term persistence and long-term effects in living
organisms, they are ranked among the most toxic chemical substances in the envi-
ronment. Their presence in food poses a serious threat to the health and life of
consumers. One of the protection measures against pesticide residues in food is to
determine the maximum residue levels permitted in a food product, and to carry out an
obligatory and systematic analytical testing of food for pesticide residue. For each
pesticide used in food production, legislators specify the definition of residue, which
enables the assessment of the health hazard. In Europe, maximum residue levels
(MRL) of pesticides permitted in food and feed, including fruits and vegetables,
2140 A. Stachniuk

intended for human consumption were established by Regulation (EC) No 396/2005

of the European Parliament and Council on pesticide residues [3].
Recently we have seen growing public concern and scientific investigations
related to the presence and control of pesticide residues in plant products to assess
the potential health hazards more thoroughly. Before food products can enter a
particular market, requirements for MRL must be met for a variety of pesticides
[4]. Therefore, there is a need for reliable and sensitive analytical methods that are
able to quantify a large number of compounds at the low limits set out in legislation
[3]. With over a thousand compounds to monitor [5] in various matrices, multiresidue
methods using gas and liquid chromatography coupled with tandem mass spectrom-
etry have become the Gold Standard methodology for both quantification and
semiquantitative screening of food contaminants, such as pesticides [6, 7]. Nowa-
days, both liquid and gas chromatography are complementary techniques covering
the full range of pesticides [6, 8–12]. However, gas chromatography has the draw-
back of being unsuitable for a number of pesticides because of their instability and
polarity [13]. Compounds which are thermally labile, not volatile, and have not been
derivatized, can be separated by liquid chromatography [13]. Liquid chromatography
coupled with tandem mass spectrometry (LC-MS/MS) can analyze a much wider
range of contaminants than gas chromatography coupled with tandem mass spec-
trometry (GC-MS, GC-MS/MS). A general discussion of the application of GC-MS/
MS and LC-MS/MS for the analysis of different chemical classes of pesticides can be
found in the book chapter by Raina [14]. Liquid chromatography coupled with mass
spectrometry provides a large amount of information about complex mixtures and the
structure of the analyte without the need to derivatize the analyte. It also enables the
screening, confirmation, and quantification of hundreds of components with one
analysis. Its sample purity requirements are not stringent, and it enables a simulta-
neous analysis of contaminations with considerable variations of polarity [13].
Indeed, to achieve a wide scope and better sensitivity for a wide range of new
generation pesticides and their metabolites, which are more polar compounds [6], it
is not surprising that LC-MS/MS has become the method of choice for routine
pesticides multi-residue analysis in vegetables and fruits.
This chapter presents general information about pesticides and modern methods
of their identification by means of LC-MS/MS. A review and assessment was carried
out of the existing techniques and methods of sample preparation, separation of
multicomponent mixtures, as well as kinds of detection used in the analysis of
pesticides in fruits and vegetables by means of LC-MS/MS. Special attention is
paid to the QuEChERS technique and the triple quadrupole analyzer (QQQ) most
frequently used in MS detection. Furthermore, pesticide residues most frequently
occurring in fruits and vegetables are presented.

2 Pesticides: General Information

Pesticides have been widely used around the world since the middle of the twentieth
century [8]. About 1000 active ingredients have been employed and are currently
formulated in thousands of different commercial products [5]. They are part of a
72 LC-MS/MS Determination of Pesticide Residues in Fruits and Vegetables 2141

large group of compounds with extremely diverse physicochemical properties and

large differences with regard to polarity, volatility, and persistence [15]. They are
widely used in the control or prevention plant crop diseases. Their extensive
applications go beyond agriculture. Pesticides are used to reduce the number of
plant and animal parasites, combat plant diseases, regulate the growth and develop-
ment of plants, remove weeds, and reduce losses in food during storage and
transport. Furthermore, they help increase personal hygiene by destroying domestic
insects (fleas, lice, and cockroaches) and insects bearing diseases hazardous to
humans such as malaria, the plague, or spotted fever [16]. Their introduction
contributed significantly to increased productivity in agriculture through the stabili-
zation of agricultural crop volume and quality. Alongside unquestionable benefits,
the common use of pesticides also has an adverse impact on the natural environment,
including human and animal’s health and life, mainly because of the varying
persistence, toxicity, bioaccumulation capacity, and mobility of pesticides [17–21].
Pesticides are classified as chemicals with a high level of toxicological hazard. They
have a carcinogenic, teratogenic, and embryotoxic effects [20]. Several studies have
found that human exposure to pesticides underlies many disorders such as
Alzheimer’s disease [22], infant mortality [23], carcinogenicity [24], neurotoxicity
[25], reproductive toxicity [26], and metabolic toxicity [27]. Pesticides have an
effect on both harmful and useful organisms. Their benefits and hazards are sum-
marized in Fig. 1. The influence of pesticides on the natural environment and food
quality was described extensively in studies by Aktar [28] and Fenik [15].
The great diversity of compound structure and broad spectrum of effects and
applications makes a clear classification of pesticides difficult. The following criteria
are used most frequently: purpose of application, chemical structure, toxicity, envi-
ronmental stability, and pathways along which they penetrate target organisms [15,
16]. The classification by purpose of application is presented in Table 1. In terms of

Fig. 1 The effects of using

pesticides: their benefits and
hazards Benefits Hazards
• Improved productivity • Direct impact on humans:
• Prevention of crop • Possible carcinogenicity,
losses/yield reduction neurotoxicity, reproductive
• Disease and epidemics and metabolic toxicity of
control pesticides accumulating in
• Food quality the body;
• Reduction of food losses • Impact on environment:
during storage and • Surface water
transport contamination
• Destruction of domestic • Ground water
insects contamination
• Extended use of roads, • Soil contamination
railway lines and airports • Effect on soil fertility
thanks to the destruction of • Air contamination
weeds • Non-target vegetation
contamination
• Destruction of useful
organisms
2142 A. Stachniuk

Table 1 Classification of pesticides based on their application [16]

Type Purpose of application Type Purpose of application
Zoocides Used to exterminate Fungicides Control or kill fungi
harmful vertebrates
Insecticides Control or kill insects Herbicides Kill or inhibit the growth of
Rodenticides Control mice and other unwanted plants
rodents
Molluscicides Control slugs, snails and Plant growth Alter the growth of plants
other mollusks regulatory
Disinfectants Control germs and
microorganisms
Bactericides Control or kill bacteria Defoliants Cause plants to drop their leaves
Algicides Control or kill algae Desiccants Are used to dry up living plant
tissues
Virucides Control or kill viruses Fumigants Produce gas or vapor intended to
destroy pests
Acaricides Control or kill mites Repellents Repel unwanted pests
Nematocides Kill roundworms Pheromones Used to attract insects or disrupt
their mating behavior
Ovicides Control eggs of insects Synergists Make certain pesticides more
and mites effective

chemical structure, pesticides are divided into organic and inorganic. Inorganic
pesticides are used very rarely nowadays. Arsenic insecticides, fluoride insecticides,
inorganic fungicides, and inorganic herbicides are examples of typical inorganic
pesticides [15]. Organic pesticides are an extremely numerous group of chemical
compounds, among which the following pesticides occur most often in fruits and
vegetables: organophosphorus pesticides (OPPs), organochlorine pesticides, carba-
mate pesticides, synthetic pyrethroids, triazoles, triazines, imidazoles, strobilurin,
and neonicotinoid pesticides [15, 29–31]. The most widespread food contaminants
include organophosphorus pesticides (OPPs) (e.g., dichlorvos, methamidofos, mal-
athion, methyl parathion, chlorpyrifos, diazinon, phosalone, and monocrotophos).
They include all organic compounds containing phosphorus [15]. They are used
mainly as insecticides but also as herbicides, fungicides, and acaricides. Their
presence has been confirmed in a wide variety of fruits and vegetables such as
oranges [31], apples and pears [30], bananas [30], peaches [32], grapes [10], olives
[9], cauliflowers [11], cabbages [33], celeries and peppers [34], eggplants [35],
potatoes and tomatoes [11].
The overview above mentions only the most frequently encountered groups of
chemical compounds contaminating food, and encompasses just a fragment of the
list of all pesticides permitted for use. In the literature on the subject, one can find
many extensive monographic studies containing the basic definitions, bases for
classification, ranges of toxicity, and persistence in the environment [16–20, 28,
36]. It should be remembered that, along with the progress of science and technol-
ogy, each year encounters more and more active compounds belonging to various
chemical groups permitted for use as pesticides.
72 LC-MS/MS Determination of Pesticide Residues in Fruits and Vegetables 2143

3 Pesticide Legislation

In the European Union (EU), there are a number of pieces of legislation which
regulate the presence of pesticide residues in food, including fruits and vegetables.
The most important ones include Regulation EC/396/2005 that came into force on 1
September 2008 and defines a fully harmonized set of rules for pesticide residues for
all EU countries [3]. The European Commission sets the maximum permitted levels
of pesticide residues to ensure that they do not pose an unacceptable health risk for
consumers. This regulation simplifies the previously existing legislation by setting
European Maximum Residue Levels (MRLs). Until 5 January 2018, 120 amend-
ments were made to the regulation (the first one on 22 February 2006 and the last one
of 4 January 2018) [3]. The list of the MRLs for all the various commodities is easily
accessible in the EU Pesticides database [37]. Commission Decision 2002/657/EC
implementing Council Directive 96/23/EC concerning the performance of analytical
methods and the interpretation of results [38] is another extremely important piece of
legislation that must be observed by laboratories where food is analyzed by means of
LC-MS/MS. This document includes definitions and descriptions of how to assess
trueness, recovery, repeatability, ruggedness, as well as detailed requirements for MS
detection and identification of targeted substances. This piece of legislation is critical
in the context of the required number of identification points corresponding to the
accurate number of precursor ion–product ion transitions, guaranteeing the reliabil-
ity of the analytical method based on MS detection. Nowadays, many laboratories all
over the world develop and validate their own methods for pesticide residue analysis
because, depending on the analytical technique chosen, different approaches for
sample treatment can be considered. Therefore, it is extremely important for the
newly developed methods to be reliable. The European Commission’s Directorate
for Health and Food Safety (DG SANTE) provides a guidance document [39] on
analytical quality control (SANCO) to laboratories for the validation of methods for
pesticide residue analysis in food and feed. Laboratories performing pesticide
residue analyses should meet the performance requirements detailed in the
SANCO Guidance [39]. An overview of the identification criteria for LC-MS/MS
in various application areas are summarized by Mol et al. [40].
The regulatory frameworks require each chemist analyst dealing with the analysis
of pesticides in food to have an excellent knowledge of the current law and to apply it
while carrying out LC-MS/MS analyses in the laboratory, particularly when new
detection methods are being developed.

4 Approach to Pesticide Identification by Means of

LC-MS/MS

In pesticide residue analysis, similarly to the analysis of other food contaminants,

there is a clear tendency to prepare multiresidue methods that enable the determina-
tion of hundreds of analytes in single analysis [5, 13, 41, 42]. This approach has
become a necessity because of the use of diverse active substances in agricultural
2144 A. Stachniuk

pesticides, which can lead to the cooccurrence of a wide range of pesticides with
different chemical properties in food. A growing need for such analyses is also a
natural consequence of introducing low maximum pesticide residue levels in the
European Union and their obligatory control. The increased awareness of consumers
who demand products that are safe and free from chemical contaminants is also
significant.
Multiresidue methods follow the basic analytical steps including sample prepa-
ration (homogenization, extraction, and cleanup) and determination (separation and
detection).

4.1 Sample Preparation

Sample preparation is the cornerstone of any effective multiresidue method. The

correct extraction and preparation of the sample are of fundamental importance
because they determine the quality and reliability of the determination result.
The choice of the sample preparation procedure is determined by the kind of
foodstuff and the chemical structure of the determined contaminants. Many
different extraction procedures are used in laboratories [41, 43–45]. So far, to
purify and extract organic compounds from fruits and vegetable samples, various
preparation techniques have been employed, such as liquid–liquid extraction
(LLE) [46], microwave-assisted extraction (MAE) [47], accelerated solvent
extraction (ASE) [48], matrix solid-phase dispersion (MSPD) [49], solid-phase
extraction (SPE) [50], solid-phase microextraction (SPME) [51], supercritical
fluid extraction (SFE) [52], and membrane extraction techniques [53]. Advan-
tages and disadvantages of these techniques have been discussed extensively in
many papers [43–45]. The majority of the aforementioned methods are rather
time-consuming, labor-intensive, complicated, and expensive, and they consume
a large volume of solvent. Therefore, they fail to meet the standards of a
multiresidue application [30]. Nowadays, there is a marked trend to have simple,
fast, easy-to-perform procedures [54]. One example of such a procedure is the
so-called Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method
of pesticide analysis. This technique involves liquid–liquid partitioning using
acetonitrile and purifying the extracts using dispersive solid-phase extractions.
Since its development and publication [55], the QuEChERS method has been
gaining significant popularity. According to Web of Science (January 2018),
over 1200 papers on using the QuEChERS method for pesticide analysis in
food have been already published. Now, it is the method of choice for food
analysis because it combines several steps and extends the range of pesticides
recovered in comparison with older, more difficult, and laborious extraction
techniques (Fig. 2).
The QuEChERS procedure was developed in the years 2000–2002, and for the
first time it was presented at the 4th European Pesticide Residue Workshop in Rome
in 2002 by Anastassiades et al. [56]; it was published a year later [55]. It was used as
a method to prepare samples for the determination of pesticide residues in fruits and
72 LC-MS/MS Determination of Pesticide Residues in Fruits and Vegetables 2145

QuECheRS

Add solvent Add QuECheRS salt

liquid-liquid
extraction (LLE),
microwave-assisted
extraction (MAE),
accelerated solvent
extraction (ASE),
Reported matrix solid-phase LEADER Centrifuged
techniques dispersion (MSPD), technique
solid-phase
extraction (SPE), Homogenate
solid-phase
microextraction
(SPME), LC-MS dSPE tube
supercritical fluid analysis
extraction (SFE)
membrane extraction
techniques
Centrifuged

Fig. 2 Extraction techniques used to isolate pesticides from food by means of the LC-MS/MS method

vegetables by means of GC-MS [55]. Its first application in conjunction with LC-MS
methods was recorded in 2005 as a continuation of research conducted by
Anastassiades in 2003 [57]. The results obtained then from the validation method
for over 200 different pesticides in a dozen or so fruits and vegetable samples with a
varied matrix composition attracted an enormous interest in the procedure and led to
its very fast growth. The basic concept of QuEChERS [55], which was developed to
determine pesticide residues in fruits and vegetable samples with a high water
content, is based on extraction by centrifugation of the food matrix with acetonitrile.
Water is separated from acetonitrile through the addition of adequately mixed salts
(anhydrous magnesium sulfate and sodium chloride). The extract is then cleaned up
using dispersive solid-phase extractions (d-SPE) with anhydrous magnesium sulfate
and primary-secondary amine (PSA), which efficiently removes many polar inter-
fering substances present in the matrix.
The QuEChERS procedure [55] consists of several steps [54]. The first step is to
weigh 10 g of the homogenized sample into a 50 mL polypropylene tube, then add
10 ml of acetonitrile and shake the sample vigorously for 1 min. After that, an
internal standard (ISTD) is added and the tube is shaken intensively for another
1 min by hand. Next, an addition of salt mixture (4 g anhydrous magnesium sulfate
and 1 g sodium chloride) is followed by intense agitation. The whole sample is again
shaken vigorously for 1 min and centrifuged. Afterwards, 1 mL of organic superna-
tant (upper acetonitrile layer) is transferred into dispersive solid phase extraction
(d-SPE) tubes containing 150 mg of anhydrous magnesium and 50 mg of PSA. Then
the sample is shaken vigorously for 1 min and centrifuged. The obtained supernatant
2146 A. Stachniuk

is transferred to an autosampler vial for LC-MS/MS analysis. The schematic flow

chart for the main steps is presented in Fig. 2.
Depending on the objective of the analysis, the type of analysed compounds, kind
and composition of the matrix, the original procedure [55] is subjected to various
modifications. To apply the QuEChERS method to a wide range of pesticides,
including pH-sensitive pesticides, the original procedure was modified with buffers.
Lehotay et al. modified the method to use a relatively strong acetate as a buffer and
the method became the AOAC Official Method 2007.01 [58]. Annastassiades et al.
chose citrate as the buffer and this version was named by the European Committee
for Standardization (CEN) as Standard Method EN 15662 [59]. Other frequent
modifications include: changes in the composition and/or proportion of the salts
used in extraction [60], changes in the composition and/or proportion of the sorbents
used [10, 12, 61], using various proportions of buffers: citrate [12, 32, 62, 63] or
acetate [12, 32, 63] and (in order to maintain the optimum pH of the sample within
the 5–5.5 range) using appropriate acids [29, 60] or bases [29]. One should also note
other variants of the method that are based on omitting the cleanup step completely
[40], using the sorbent deposit in the form of a column [64], replacing acetonitrile
with ethyl acetate as the organic solvent [65], or introducing an additional step where
the analytes are concentrated in the cleaned up extract by evaporating and/or
replacing the solvent [61, 66, 67]. Lee et al. proposed an interesting modification
of the method [68]. To improve the recovery of some problematic highly
polar pesticides in pepper (Capsicum), they introduced the dry ice-partitioning
QuEChERS method. This approach uses dry ice to separate a sample extract into
the acetonitrile layer and aqueous layer without the need for salting out and centri-
fugation [68]. The protocol developed involves two extraction methods. The
first detects the acetonitrile layer for general pesticides, and the second combines
the acetonitrile and the aqueous layer for propamocarb, pymetrozine, and flonicamid
metabolites [68].
The selection of the right sorbent in sample cleanup is a key stage of the
QuEChERS procedure. In dispersive solid-phase extractions (d-SPE) used in
QuEChERS, the traditional sorbent deposit was replaced with loose sorbent, added
directly to the sample solution. The method involves the d-SPE cleanup procedure
whereby the extract is shaken with a small amount of sorbents and MgSO4 [45].
Among the different kinds of d-SPE sorbents, primary secondary amine (PSA) is the
most commonly used in the QuEChERS method; it is used to remove polar organic
acid, fatty acids, polar pigments, and some sugar [69]. In some modified QuEChERS
methods, graphitized carbon black (GCB) and octadecyl silica (C18) are applied to
remove sterols, pigments, and nonpolar interfering substances [69]. GCB is useful
for the purification of carotinoids, chlorophyll, and sterols with the disadvantage of
losses of planar analytes [54]. C18 provides good results in the purification of
samples with significant fat and wax content but recoveries of the more lipophilic
pesticides may suffer [54]. One of the new sorbents recently used in the QuEChERS
method are carbon nanotubes (CNTs). Based on the principle of carbon atom layers
in the wall of nanotubes, the carbon nanotubes are classified as multiwalled carbon
nanotubes (MWCNTs) and single-walled carbon nanotubes (SWCNTs) [69–72].
72 LC-MS/MS Determination of Pesticide Residues in Fruits and Vegetables 2147

MWCNTs exhibit a good affinity for polycyclic compounds and could be used as
effective d-SPE sorbents to adsorb the interfering substances in fruits and vegetable
matrices owing to their unique structure and huge surface area [69]. They have been
used for the cleanup of complex matrices such as grape, mango juice, wheat,
spinach, carrot, apple, citrus, peanut, leek, and leaf lettuce [10, 50, 69, 71]. The
application of different d-SPE sorbents for the QuEChERS cleanup step of various
matrix types is extensively described by Rajczak and Tuzimski [54].
The QuEChERS extraction technique makes it possible to reduce the number of
the necessary steps of analysis, reagents, and laboratory glassware. It also simplifies
the step of analyte extraction and extract cleanup. In comparison with other extrac-
tion techniques, it is characterized by very good results in removing matrix ingredi-
ents and high recovery of the target compounds [45, 54, 69]. The QuEChERS
approach is also in accordance with the principles of green chemistry due to low
solvent consumption and absence of chlorinated solvents and very small generation
of waste [54]. Since the introduction of the QuEChERS method, the literature on the
subject has presented numerous examples of its use to determine pesticides in a
whole range of food products [12, 73] as well as studies reviewing the method [44,
54, 73] and comparing it with other extraction techniques [12, 45].

4.2 Sample Determination

The accurate determination of pesticides in complex matrices such as fruits and

vegetables is a challenge due to the complexity of the matrix and low mass fraction
levels to be dealt with. The choice of appropriate separation and detection techniques
is a step of fundamental importance.
Most multiresidue methods (MRMs) reported for fruits and vegetables are based
on the use of liquid chromatography coupled with tandem mass spectrometry
(LC-MS/MS), which is a powerful technique that provides excellent selectivity
and sensitivity, allowing a reliable identification and quantification of a wide range
of pesticides and their metabolites [42]. The most important advantages of coupling
LC with MS/MS include: a clear identification of sample components, very high
sensitivity of the analytical system, possibility of identifying all or most of the
sample components, high repeatability of measurements, possibility of quantitative
analysis (generating real chromatographic peaks by the LC system), possibility of
distinguishing substances with identical molecular mass but different retention times
(isomers), and possibility of obtaining additional information about each sample
component by means of fragmentation [42, 74, 75]. Using multiresidue methods in
chromatographic determinations, the problem of the coelution of sample compo-
nents occurs. Coupling liquid chromatography with mass spectrometry allows the
identification of the studied compound based on the mass spectrum, enabling the
quantification of substances even if they are not fully chromatographically separated
[74]. This technique provides information regarding the characteristic ion of each
analyte as well as two or more transitions of these ions, which is useful for
2148 A. Stachniuk

LC MS

HPLC source
analyzers
or of detector
m/z
UHPLC ions

Fig. 3 Block diagram of the LC-MS/MS system

quantifying and confirming analytes at concentrations consistent with the maximum

residue levels (MRLs) established [76].
In a typical LC-MS/MS system, the studied sample is first separated in the LC
system and its consecutive fractions flowing out of the chromatographic column are
subjected to ionization and introduced into the spectrometer. The typical LC-MS/MS
system is shown in Fig. 3.
Liquid chromatography is one of the most widespread methods of separating
complex samples. It is used for the analysis polar and/or nonvolatile and ther-
mally labile pesticides for which GC conditions are not suitable. Most of the
procedures used to determine pesticides in fruits and vegetables employ the
HPLC technique in a reversed phase (RP) system, using gradient elution with a
linear increase of the percentage of the organic solvent [69, 77]. The common
stationary phases are based mainly on the nonpolar octadecylsilane phase (C18)
[69, 78, 79]; the medium-polar octylsilane (C8) phase is used much more rarely
[80]. The mobile phase consists of a mixture of acetonitrile, methanol, and water
in various volume ratios [40, 60, 78]. In order to improve the ionization capacity,
the use of different additives to the mobile phases has been described such as
organic acid (formic [69, 78] and acetic [34]), ammonium salts (ammonium
formate [60, 77, 78] or ammonium acetate [33, 67]) or a combination of an
organic acid with its ammonium salt [77, 79]. One of the crucial technological
revolutions in LC was the implementation of ultrahigh pressure liquid chroma-
tography (UHPLC). The particle size of the solid phase was reduced from 5 μm to
sub-2 μm resulting in an enhanced resolution in a shorter runtime [81]. The use of
short columns with the particle diameter not larger than 2 μm was first proposed
by Waters in 2004 [82]. The UHPLC system is currently the optimum solution for
the separation of multicomponent mixtures.
Tandem mass spectrometry (MS/MS) is a key analytical technique enabling the
identification of pesticides in different kinds of fruits and vegetables. The basic
principle of MS/MS is the selection of the precursor ion, fragmentation of this ion,
usually by collision-induced dissociation (CID), and measurement of the m/z ratio of
the product ion formed [75]. Ionization in LC-MS/MS is mainly based on atmo-
spheric pressure ionization (API), which enables the introduction of considerable
72 LC-MS/MS Determination of Pesticide Residues in Fruits and Vegetables 2149

volumes of solutions flowing out of the chromatographic column into the mass
spectrometer without the risk of losing a high vacuum [83]. The analyzers and
detector of the mass spectrometer are kept in a high vacuum to avoid accidental
collisions with air molecules [13]. The most frequently used ionization methods
in pesticide determination include electrospray ionization (ESI) and atmospheric
pressure chemical ionization (APCI) [7, 13]. However, APCI appears to have
been overshadowed by the extreme popularity of ESI [7]. A discussion about the
application of both of these ionization methods in the LC-MS/MS analysis of
pesticides can be found in our previous paper [13]. The analyzer is the most
important part determining the parameters of a spectrometer. Tandem mass
spectrometry (MS/MS) is a coupled system of two analyzers of the same type
or different types, characterized by a high separation efficiency [13]. The current
trend in food control is the use of tandem MS such as a triple quadrupole (QQQ)
as well as high-resolution mass spectrometers (HRMS) such as quadrupole-time
of flight (Q-TOF) and quadrupole-linear ion trap (Q-Trap). Among all of the
above, the triple quadrupole (QQQ) is the most common MS analyzer in LC-
based methods for multiresidue analysis in food nowadays. This is because of
their excellent quantification and identification properties for a group of target
compounds [7, 41]. As has been described previously by Stachniuk and Fornal
[13], the conventional QQQ instrument is a serial connection of quadrupole
analyzers, the middle of which performs the role of the collision cell. The first
quadrupole only allows the so-called precursor ions (ions of a specific m/z value)
to pass through. The precursor ions selected in the first analyzer are fragmented in
the collision cell, filled with neutral gas and constituting the second quadrupole.
The fragmentation spectrum, i.e., the m/z values of product ions, is recorded in
the detector. When the third quadrupole allows only the selected product ions, the
spectrometer works in the multiple reaction monitoring mode (MRM). When the
third quadrupole allows all product irons, it works in the scan mode (full mass
spectrum collection). Working in the scan mode, a tandem system of the QQQ
type operates like a classic quadrupole analyzer. When measuring the full spec-
trum, the first two quadrupoles function as beam collimators, and the entire
analysis is performed in the third quadrupole [13]. Thus, a mass spectrometer
of the QQQ type can work with different settings for ion analysis parameters.
First, MS can work in the positive or negative ion detection mode. Second,
several ways of ion detection are possible. The first one is based on the moni-
toring of the total ion current (TIC) from the preset m/z range. The second one is
based on monitoring only selected ions (SIM) characteristic of the compounds
searched for. The third one, multiple reaction monitoring (MRM) (also called
selected reaction monitoring (SRM)), enables the confirmation of the presence of
fragmented ions formed from a specific precursor ion [13]. QQQ analyzers are
used mainly for quantification and confirmation purposes. Typically, two or three
MRM transitions are selected for a targeted analysis of pesticide residues: one for
quantification and an additional one for confirmation purposes [62].
The use of the MRM mode in QQQ instruments for the determination of pesticide
residue in food seems to have certain limitations: they require acquisition parameter
2150 A. Stachniuk

optimization for each compound analyzed, the number of the analyzed compounds is
limited, only compounds from the target list can be detected, and retrospective data
analysis is impossible [78]. Therefore, there has been a move to an alternative
approach using HRMS capable of providing full spectrum data. Operating in a full
scan mode, HRMS offer a significantly higher analysis selectivity and can detect an
unlimited number of compounds. HRMS analyzers used for pesticide residue anal-
ysis in fruits and vegetables are the Q-TOF [84] and Q-Trap [78]. However, only few
papers have described the use of HRMS for quantitative purpose [6, 11, 66, 78, 84].
The triple quadrupole remains the most common instrument in quantitative target
analysis. Several reviews on the subject help to interpret the trends within the field
[7, 13, 41, 42, 85]. The current LC-MS/MS strategies for multiresidue methods are
summarized in Table 2.
One of the most limiting factors in LC-MS/MS pesticide residue analysis is the
occurrence of the matrix effect. The ionization of target analytes can be affected
when competition between the analyte and coeluting components for the available
charge occurs. Exo- and endogenic substances present in the matrix can interfere
with the analyte in the ion source [13]. This effect may cause the suppression or
enhancement of ionization, leading to quantification errors. In various review arti-
cles, different approaches have been discussed to overcome this analytical problem
[13, 41, 102, 103]. As reported by Ucles et al. [103], several approaches are used to
eliminate or reduce matrix effects; they include optimizing sample preparation to
remove interfering compounds from the samples [104], changing LC conditions to
avoid the coelution of analytes and interfering compounds [105], changing MS
conditions to reduce the occurrence of the matrix effect in the ion source [106],
dilution of the sample [106] or using chemical treatment measures [107] or calibra-
tion techniques such as matrix-matched calibrations [29], and the standard addition
method [13].
To reduce the matrix effect and increase the accuracy of determination during the
sample preparation step in multiresidue analysis of pesticides in a wide range of food
matrices, matrix-matched calibrations with the addition of a single internal standard
are mainly used.

5 The Most Frequently Detected Pesticides in Fruits and

Vegetables

Based on the review of the literature (Table 2), it was determined how frequently
pesticide residues, determined by means of LC-MS/MS, occur in fruits and vegeta-
bles. The obtained results are presented in Fig. 4.
The most frequently detected pesticide residues in the fruit group included:
carbendazim, azoxystrobin, chlorpyrifos, imazalil, imidacloprid, acephate,
acetamiprid, cyprodinil, dimethoate, cypermethrin, metalaxyl, methomyl, and
thiabendazole. As Fig. 4 indicates, carbendazim is one of the main detectable
residues in fruit, and occurred in the concentration range of 0.0026–3.4 mg kg 1
as reported in eight different papers. The residues of carbendazim detected in
72 LC-MS/MS Determination of Pesticide Residues in Fruits and Vegetables 2151

Table 2 Current LC-MS/MS strategies for the analysis of pesticide residues in fruits and vegeta-
bles (2017–2010)
No. of Extraction Detector Ionization MS
No. Matrix analytes methods MS methods mode Refs.
1 Mango 68 modified Q-TOF ESI scan, [84]
QuEChERS EIC
2. Goji berry 8 QuEChERS QQQ ESI MRM [86]
3. Fruits and 60 QuEChERS QQQ ESI MRM [29]
vegetables
4. Cabbage 7 QuEChERS QQQ ESI SRM [33]
5. Spring onion 5 QuEChERS QQQ ESI MRM [87]
6. Tomato 109 QuEChERS QQQ ESI MRM [60]
7. Oranges 115 QuEChERS QQQ ESI MRM [31]
8. Peach, grapes, 17 QuEChERS QQQ ESI MRM [30]
bananas,
apples, pears,
strawberries
9. Tropical fruits 30 QuEChERS QQQ ESI MRM [77]
10. Grape and 41 QuEChERS Q-trap APPI MRM [10]
mango juices
11. Tomato 57 QuEChERS QQQ ESI MRM [88]
12. Leek, leaf 70 Modified QQQ ESI SRM [69]
lettuce, QuEChERS
13. Soya 10 QuEChERS QQQ ESI MRM [61]
14. Apple, 120 QuEChERS QQQ ESI SRM [62]
cucumber
15. Fruits and 60 Modified TOF ESI Scan, [11]
vegetables QuEChERS EIC
16. Fruits and 100 LLE Q-TOF ESI Scan, [6]
vegetables EIC
17. Fruits and 186 QuEChERS QQQ ESI MRM [89]
vegetables
18. Apple, guava, 46 Modified QQQ ESI MRM [65]
kaki, peach QuEChERS
19. Fruits and 199 QuEChERS Q-TOF ESI Scan, [66]
vegetables EIC
20. Bananas 128 QuEChERS QQQ ESI MRM [90]
21. Olives 90 QuEChERS QQQ ESI MRM [9]
22. Fruits and 6 LLE QQQ ESI, SRM [91]
vegetables APCI
23. Fruits and 100 QuEChERS QQQ ESI SRM [34]
vegetables
24. Vegetables 44 QuEChERS QQQ ESI MRM [92]
25. Fruits 29 LLE Q-TOF ESI MRM [93]
26. Fruits and 17 QuEChERS QQQ ESI, MRM [94]
vegetables APCI
27. Vegetables 18 QuEChERS Q-trap ESI MRM [35]
(continued)
2152 A. Stachniuk

Table 2 (continued)
No. of Extraction Detector Ionization MS
No. Matrix analytes methods MS methods mode Refs.
28. Cucumber, 5 QuEChERS QQQ ESI MRM [81]
tomato, apple,
lettuce, grape
29. Fruits and 150 QuEChERS QQQ ESI SRM [80]
vegetables
30. Fruits and 20 Modified QQQ ESI MRM [95]
vegetables QuEChERS
31. Paprika 168 Modified QQQ ESI MRM [68]
QuEChERS
32. Fruits and 54 LEE QQQ ESI SRM [46]
vegetables
33. Fruits and 32 QuEChERS QQQ ESI MRM [12]
vegetables
34. Fruits and 14 QuEChERS QQQ ESI SRM [96]
vegetables
35. Onion 5 MSPD QQQ ESI MRM [49]
36. Olives 104 (I) QQQ ESI MRM [97]
QuEChERS,
(ii)MSPD
37. Eggplant, 10 (I) QQQ ESI MRM [98]
citrus fruits QuEChERS,
(ii)
CHEMAC,
(iii)STEMIT
38. Fruits and 11 LLE QQQ, ESI SRM [99]
vegetables Q-TOF
39. Grape 150 QuEChERS Q-trap ESI MRM [100]
40. Fruits and 69 QuEChERS QQQ ESI MRM [101]
vegetables

fruit samples are summarized in Table 3. The highest level of carbendazim

(3.4 mg kg 1) was recorded in a papaya sample [77].
The most frequently detected pesticide residues in the vegetables group
included: acetamiprid, imidacloprid, carbendazim, azoxystrobin, chlorpyrifos,
acephate, cypermethrin, dimethoate, metalaxyl, cyprodinil, imazalil, methomyl,
and thiabendazole. The presence of the most frequently occurring acetamiprid
was also confirmed by eight authors as was the case with fruits (Fig. 4). The results
of the survey for acetamiprid are presented in Table 4. Monitoring data showed that
the mean acetamiprid concentrations varied from 0.01 to 0.86 mg kg 1. The highest
acetamiprid level was found in a lettuce sample with the concentration of
0.86 mg kg 1 [89].
The results shown in Tables 3 and 4 were compared with the current maximum
residue levels permitted by the European Union as of 5 January 2018 [3]. It should
72 LC-MS/MS Determination of Pesticide Residues in Fruits and Vegetables 2153

Fig. 4 The frequency of the 9

most commonly detected
8
pesticides in fruits and
vegetables, based on the 7
reviewed papers from Table 1 6
5
4
3
2
1
0

FRUITS VEGETABLES

Table 3 Residues of carbendazim in fruit samples, based on the reviewed papers from Table 1
MRLa Range min–max
Matrix Total Positive (mg kg 1) (mg kg 1) Refs.
Tamarillo No 1 0.1 ~0.05 [93]
define
Lulo 2 2 0.1 0.019–1.34 [77]
Carambolo 2 1 0.1 0.0021
Granadilla 2 1 0.1 0.66
Mangostan 2 1 0.1 0.0035
Tamorillo 2 2 0.1 0.08–0.29
Gulupa 2 2 0.1 0.0026–0.0072
Maracuya 2 2 0.1 0.03–0.21
Papaya 2 1 0.2 3.4
Feijoa 2 1 0.1 0.0032
Citrus 120 5 0.1 0.01–0.02 [98]
Peaches No No 0.2 0.02–0.178 [30]
define define
Apple No 1 0.2 0.088
define
Black 36 9 0.1 0.07–0.51 [29]
currant
Red currant 32 5 0.1 0.06–0.13
Banana 128 1 0.1 0.024 [90]
Apple 32 No 0.2 1.304–1.325 [11]
define
Guava 12 No 0.1 0.011–1.445
define
(continued)
2154 A. Stachniuk

Table 3 (continued)
MRLa Range min–max
Matrix Total Positive (mg kg 1) (mg kg 1) Refs.
Apple 64 4 0.2 0.02–0.09 [89]
Apricot 9 2 0.2 0.04–0.1
Banana 17 1 0.1 0.63
Cherry 16 5 0.5 0.011–0.068
Grape 72 9 0.3 0.03–0.215
Kiwi 5 1 0.1 0.03
Lemon 28 6 0.7 0.014–0.334
Orange 29 3 0.2 0.01–0.124
Peaches 20 3 0.2 0.024–0.151
Pear 22 1 0.2 0.235
Strawberry 14 1 0.1 0.02
a
The current maximum residue levels permitted by the European Union as of 5 January 2018 [3]

Table 4 Residues of acetamiprid in vegetables samples, based on the reviewed papers from Table 1
Matrix Total Positive MRLa (mg kg 1) Range min-max (mg kg 1) Refs.
Arugula 11 1 3.0 0.102 [89]
Aubergine 26 11 0.2 0.018–0.23
Bean 15 1 0.15 0.01
Cucumber 49 9 0.3 0.016–0.069
Lettuce 29 6 3.0 0.01–0.86
Mushroom 58 1 0.01 0.017
Pepper 59 29 0.3 0.01–0.79
Purslane 5 3 3.0 0.028–0.08
Tomato 118 58 0.5 0.01–0.29
Zucchini 8 5 0.3 0.02–0.241
Green pepper 2 1 0.1 0.024 [34]
Broccoli 8 2 0.4 0.014–0.023 [29]
Green chili 1 1 0.3 0.015 [92]
Paprika 50 1 0.3 <MRL [68]
Tomato 60 6 0.5 0.0–0.03 [88]
Tomato 1471 1 0.5 0.03 [101]
Tomato 345 23 0.5 0.015–0.37 [60]
a
The current maximum residue levels permitted by the European Union as of 5 January 2018 [3]

be remembered that these levels are constantly updated, and different levels may
apply to a fruit or vegetable depending on the date of the analysis. The changes of the
maximum residue levels from since the time Regulation (EC) No 396/2005 came
into force, using the example of carbendazim in orange and lemon samples as well as
acetamiprid in broccoli and tomato samples, are presented in Tables 5 and 6,
respectively.
72 LC-MS/MS Determination of Pesticide Residues in Fruits and Vegetables 2155

Table 5 Evolution of MRLs for carbendazim in orange and lemon [37]

Reg. (EU) No 559/ Reg.(EU) No 893/ Reg. (EU) No 839/ Reg. (EU) No 149/
2011 2010 2008 2008
Lemon 0.7 0.7 0.5 0.5
Orange 0.2 0.5 0.5 0.5

Table 6 Evolution of MRLs for acetamiprid in broccoli and tomato [37]

Reg. (EU) Reg.(EU) Reg. (EU) Reg. (EU) Reg. (EU) Reg. (EU) Reg. (EU)
No 625/ No 1902/ No 401/ No 500/ No 978/ No 508/ No 149/
2017 2016 2015 2013 2011 2011 2008
Broccoli 0.4 0.4 0.4 0.4 0.3 0.01 0.01
Tomato 0.5 0.5 0.2 0.2 0.15 0.1 0.1

The differences of the results obtained in various papers suggest that further and
continuous investigation of pesticide residues in fruits and vegetables is necessary to
ensure consumer safety.

6 Conclusions

The analysis of pesticide residues in fruits and vegetables is a key issue that is
strongly related to human health. For more than a decade, a lot of progress has
been made in multiresidue LC-MS/MS methods for pesticide analysis in terms of
the ease of preparation, analysis time, and number of targeted compounds with
concomitant improvements of instruments performance. Today, high perfor-
mance liquid chromatography coupled with triple quadrupole mass spectrometry
in combination with QuEChERS extraction is the most used technique for
pesticide residue analysis in fruits and vegetables. Operating in the multiple-
reaction monitoring (MRM) mode provides a high degree of sensitivity and
selectivity, and is highly suited for quantitative identification and determination
of target analytes in samples. However, it does not allow the identification of
nontarget compounds. Therefore, the use of nontargeted screenings employing
high-resolution mass spectrometers (HRMS), e.g., quadruple-time-of-flight
(QTOF) mass analyzers, is worth considering. Both (QQQ and QTOF) are
complementary tools to develop large-scale screening methods. The conducted
review of data provided in the literature reveals a considerable variation in the
contamination of fruits and vegetables with pesticides. At the same time, it shows
that monitoring and pesticide residue determination methods for various groups
of fruits and vegetables should be continually developed and improved in order
to fully protect the health of consumers.
2156 A. Stachniuk

Acknowledgments Author gratefully acknowledges the use of the facilities and services of the
Center for Interdisciplinary Research at The John Paul II Catholic University of Lublin, Poland,
co-funded by the European Union from the European Regional Development Fund in the frame
of the Operational Programme Development of Eastern Poland 2007–2013 (POPW.01.03.00-
06-003/09-00). This work was supported by the Polish National Science Centre via the research
project DEC-2017/01/X/ST4/00722.I also acknowledge assistance in preparation of tables
provided by Ms Agata Surma.

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