Dllme

1
Current Trends in Liquid-Liquid Microextraction

for Analysis of Pesticide Residues in
Food and Water
Sara C. Cunha, J.O. Fernandes and M. Beatriz P.P. Oliveira
REQUIMTE, Department of Bromatology, Faculty of Pharmacy,
University of Porto, Rua Aníbal Cunha 164 4099-030 Porto
Portugal
1. Introduction
Since the middle of last century, the use of organic synthetic pesticides became a
widespread practice, in order to better prevent, control and destroy pests. Despite their
usefulness in the increment of food production, the extensive use of pesticides during
production, processing, storage, transport or marketing of agricultural commodities can led
to environmental contamination and to the presence of residues in food. Real and perceived
concerns about pesticide toxicity have promoted their strict regulation in order to protect
consumers, environment and also the users of pesticides. Thus, reliable and accurate
analytical methods are essential to protect human health and to support the compliance and
enforcement of laws and regulations pertaining to food safety.
The first analytical methods for pesticide analysis were developed in the years 1960s,
employing an initial extraction with acetone, followed by a partitioning step upon addition
of a non-polar solvent and salt; these methods involved complex and solvent-intensive
cleanup steps. Moreover, the instruments available for analysis of the target compounds had
a relative low selectivity and sensitivity. The development of technology and robotic in the
1990s allied to the aim to reduce manual interference and to allow sample preparation
during non-working time, has boosted the development of automated sample preparation
techniques such as supercritical fluid extraction and pressure liquid extraction. Though
initially very promising, these techniques have not succeeded in the field of pesticides
analysis for various reasons, namely high price and low reliability of the instruments, and
inability to extract different pesticide classes in foods with the same efficiency, often
requiring separate optimization for different analytes. Later, a successful simplification of
“traditional” solvent sample preparation, QuEChERS (quick, easy, cheap, effective, rugged,
and safe) was presented by Lehotay and collaborators (Anastassiades et al., 2003). This
procedure, involving a simple extraction/partition using acetonitrile and salts followed by a
simple dispersive cleanup, has been adopted for the analysis of many pesticide residues in
food (Cunha et al, 2010). Two similar QuEChERS methods achieved the status of Official
Method of the AOAC International (Lehotay, 2007) and European Committee for
Standardization (CEN) standard method EN 15662 (Standard Method EN 15662).
Unfortunately, the analysis of QuEChERS extracts in acetonitrile by GC-MS is not totally
straightforward. Several facts can occur: degradation of the GC column by the polar solvent,
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2 Pesticides - Strategies for Pesticides Analysis
vapor overload of the insert liner due to the high thermal expansion coefficient,
contamination of the system by co-extractives (Hetmanski et al., 2010), and reduced
enrichment factors.
Recently, the development of new analytical equipment, namely tandem mass
spectrometers coupled to LC and GC systems, allowed improvements in the sensitivity,
selectivity, and speed of analysis. Although the prohibitive costs of such equipments make
them unattainable to many groups working in this field. Such improvements in sensitivity
and selectivity could also be accomplished by innovative sample preparation techniques
recently introduced, most of them with the added benefit to be easy to execute, cost-
effective, and environmental friendly. Cloud point extraction, single-drop microextraction,
hollow fiber liquid phase microextraction, and dispersive liquid-liquid microextraction, are
examples of liquid-liquid microextraction techniques that have emerged in recent years in
the field of sample preparation and are being used increasingly. The major advantage of
microextractive techniques is the use of only microliters of solvents instead of several
hundred mililiters in the classical liquid-liquid extraction. In addition, due to the
compatibility of the solvents used and the low volumes involved, samples are easily
transferred to the next step of analysis, liquid or gas chromatography. The aim of this work
is to review the application of liquid-liquid microextraction techniques in the analysis of
pesticide residues in food and water and to compare its use with other well-established
sample preparation techniques. Special emphasis will be given to articles published in the
last four years. Principles, advantages and relative merits of each technique will be also
summarized and discussed.
2. Analytical tools for determination of pesticide residues in food and water

Pesticide analysis is almost invariably accomplished by means of a chromatographic
technique, either GC or LC coupled to universal (MS, MS/MS) or selective detectors (ECD,
electron-capture detector; NPD, nitrogen phosphorus detector; FPD, flame photometric
detector; UV, ultraviolet detector; and FLD, fluorimetric detector), following an adequate
sample preparation step. Regardless the type of chromatographic technique employed,
sample preparation remains as the limiting step to reach desired performance parameters,
due to the low legally established levels and the complex nature of the matrices in which the
target compounds are present typically in low amounts. As a rule, the physico-chemical
methods used to obtain a pesticide extract able to be chromatographically analyzed consist
in the extraction/isolation of the target analytes by an appropriate extraction technique
followed by some purification and concentration steps. The classical procedures are often
time consuming, laborious and environmental unfriendly, taking into account the large
volume of organic solvents usually required. Recently, as referred in the Introduction
section, new techniques have been introduced, offering consistently high enrichment factors
and consequently higher sensitivity for the analytes of interest, together with a significant
reduction of organic solvent consumption as well as extraction time. The most relevant
techniques in this field are further detailed in the following sections.
2.1 Sample preparation

2.1.1 Cloud-point extraction (CPE)
Watanabe and collaborators, introduced in 1976 cloud-point extraction (CPE), a promising
new separation and extraction technique, as an alternative to classical procedures with
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Current Trends in Liquid-Liquid Microextraction for
Analysis of Pesticide Residues in Food and Water 3
organic solvents (Paleologos et al., 2005). CPE or micelle-mediated extraction, is based on

the capacity exhibited by aqueous micellar solutions of some surfactants to form the cloud
point, or turbidity, phenomenon that occur when the solution is heated or cooled above or
below certain temperature. The temperature at which this phenomenon occurs is known as
the cloud-point temperature or micelle-mediated extraction (Carabias-Martínez et al., 2000).
Surfactants are amphiphilic molecules, which have a polar moiety (the head), hydrophilic in
nature, linked to a hydrophobic portion (the tail). In aqueous solution, and at low
concentrations, surfactant molecules are found in monomer form, although dimers and
trimers have also been detected (Paleologos et al., 2005).
When the surfactant concentration is increased above a certain threshold, called ‘‘critical
micellar concentration’’ (CMC), the surfactant molecules become dynamically associated to
form molecular aggregates of colloidal size. These aggregates, containing between 60 and - 100
monomers, are called micelles and are at equilibrium with a surfactant concentration in the
solution close to the CMC. Depending on the nature and concentration of the surfactant, as
well as on the solvent used, another series of structures may be formed, organized as inverse
micelles, microemulsions, vesicles, monolayers, or bilayers (Carabias-Martínez et al., 2000).
To date, liquid–liquid phase separation based on non-ionic or zwitterionic surfactant
micelles (i.e., CPE) are employed, while the use of charged surfactant species is still scarce
(Paleologos et al., 2005). Sanz et al. (2004) used non-ionic surfactants such as
polyoxyethylene 10 lauryl ether and oligoethylene glycolmonoalkyl ether (GenapolX-080) at
95ºC for 15 min to extract eight organophosphorus pesticide residues (chlorpyrifos,
diazinon, dimethoate, ethoprophos, malathion, methidathion, parathion methyl and
paration ethyl) from water, which were analyzed by HPLC-UV. The authors obtained a
enrichment factor of 20, recoveries between 27 and 105%, and limits of detection (LOD)
lower than 30 µg/L. In 2008, Santalad et al. presented a simple and rapid spectrophotometry
method based on acid-induced anionic surfactant micelle-mediated extraction (acid-induced
cloud-point extraction) coupled to derivatization with 2-naphthylamine-1-sulfonic acid to
determine carbaryl residues in water and vegetables. In this work, sodium dodecyl sulphate
(the extractant), was combined with 2-naphthylamine-1-sulfonic acid derivatization,
allowing the extraction at low temperature (45ºC). The proposed method showed good
analytical features with low LOD (50 µg/L), good precision with a relative standard
deviation (RSD) of 2.3%, and high recoveries when applied in samples (85%).
Notwithstanding the capacity to concentrate the analytes and the good recoveries achieved
with CPE, its application in the extraction of pesticide residues in food matrices is restricted,
in part due to the physico-chemical properties of the surfactant. As it is viscous, it cannot be
injected directly to conventional analytical instruments, so it has to be diluted with an
aqueous or organic solvent to reduce its viscosity, thus impairing the anticipated theoretical
preconcentration factors. Moreover, surfactant-bearing chromophores interfere with UV
detection by overlapping with the analyte signal. This problem can be solved by diluting the
surfactant-rich phase with an organic solvent prior to injection into the chromatographic
column, increasing the portion of organic solvent in LC mobile phases or using fluorescence
detection (Paleologos et al., 2005).
2.1.2 Single drop microextraction (SDME)

Drop-drop microextraction was first introduced, in 1996, by Liu & Dasgupta, (1996). They
extracted sodium dodecyl sulphate ion pairs by a microdrop (1.3 µL) of a water-immiscible
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organic solvent, suspended in a larger aqueous drop. At the same year, Jeannot and
Cantwell introduced a technique that they termed as solvent microextraction in which the
extraction medium was a droplet (8 µL) of 1-octanol held at the end of a Teflon rod and
suspended in a stirred aqueous sample solution. After extraction for a prescribed time, the
Teflon rod was withdrawn from the aqueous solution; the organic phase sampled with a
microsyringe and injected into a GC system. In this work, the authors also proposed
equilibrium and kinetic theories to explain this microextraction procedure. Subsequently,
the technique was changed to allow simultaneous extraction and injection of analytes, by
introducing as support a microsyringe, where the organic phase was suspended at the
needle tip (Jeannot & Cantwell, 1997) (Figure 1).
Fig. 1. Schematic illustration of direct immersion single-drop microextraction (from Xu et al.,

2007).
One advantage of SDME over other liquid extraction techniques is the small volume of
organic solvent required. Additionally, in this technique, analytes with high partition
coefficient can reach high concentrations, since they are transferred by diffusion from a
significant volume of sample (1-5 mL) to a small micro-extract (5-50 µL).
Since its introduction, different modes of SMDE have been developed, in order to improve
extraction efficiency, such as direct SDME, headspace SDME (HS-SDME) and continuous-
flow microextraction (CFME).
Direct SDME consists of suspending a microdrop of organic solvent at the tip of a syringe,
which is immersed in the aqueous sample. An alternative approach was described as
dynamic technique by He & Lee (1997), in which organic solvent repetitively forms a film
inside the syringe barrel by continuously pulling and pushing of the syringe plunger.
Extraction takes place between the sample solution and the organic film (He & Lee, 2006).
Direct SDME has extensively been used for the direct extraction of pesticide residues from
aqueous samples (Table 1). Xiao et al. (2006) evaluated two types of SDME, static and
dynamic, in extraction of six organophosphorus pesticides (OPPs) (dichlorvos, phorate,
fenitrothion, malathion, parathion, quinalphos) from water and fruit juice. Significant
parameters affecting SDME performance such as extractant solvent, solvent volume, stirring
rate, sample pH and ionic strenght were evaluated. The authors verified that static SDME
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procedure allowed an enrichment factor of the six OPPs nearly 100 fold, which were much
better than the results obtained with the dynamic mode. The optimized static SDME
procedure in conjugation with GC-FPD allowed good detection limits ranging from 0.21 to
0.56 µg/L. In the same year, Zhao et al. (2006) also optimized a SDME procedure for
extraction of seven OPPs (ethoprophos, diazinon, parathion methyl, fenitrothion, malathion,
isocarbophos and quinalphos) in orange juices with analysis under GC-FPD. An effective
extraction was achieved by suspending during 15 min a 1.6 µL drop of toluene to the tip of a
microsyringe immersed in a 5 mL donor aqueous solution with 5 % (w/v) NaCl and stirred
at 400 rpm. The seven OPPs were extracted from orange juice samples with good limits of
detection (below 5 µg/L). However, better detection limits for 13 OPPs pesticides (ranging
from 0.001 to 0.005 µg/L) in water were obtained by Ahmadi et al. (2006) using SMDE with
a modified 1.0 µL microsyringe and GC-FPD, compared to 10 µl microsyringe used in the
works above referred. By using a 1.0 µL microsyringe the repeatability of the drop volume
and the injection were improved, due to the maximum volume of microsyringe without
dead volume. On the other hand, the modification of the needle tip caused increasing cross
section of it and increasing adhesion force between needle tip and drop, thereby increasing
drop stability and allowing a higher stirrer speed (up to 1700 rpm). The method used 0.9 µl
of carbon tetrachloride as extractant solvent, 40 min of extraction time, stirring at 1300 rpm
and no salt addition. The potential of SMDE was also investigated by Liu et al. (2006) in the
extraction of four fungicides from water and wine samples. Additionally, SDME has been
applied in the extraction of organochlorine pesticides (OCPs) in various matrices (Table 1).
Qia & He (2006) introduced a funnel from SDME to extract 11 OCPs and 2 pyrethroid
pesticides from tea samples and analyze by GC-ECD. More recently, Cortada et al. (2009a)
proposed a SDME procedure comprising a 2 µL toluene microdrop exposed for 37 min to 10
mL aqueous sample without salt addition and stirred at 380 rpm to extract eight OCPs from
wastewater followed by GC-MS analysis.
Contrary to the aqueous samples, vegetable and fruits, being mostly in solid or
heterogeneous form do not allow direct extraction with SDME. However, it is possible to
use SDME after a previous pretreatment. Nine OCPs ( -, -, -, σ- BHC, dicofol, dieldrin,
DDD, DDE, and DDT) were extracted with SDME from fresh vegetable (cabbage,
cauliflower, Chinese cabbage) after an adequate mixture of sample aliquots with acetone
using a ultra-sonic vibrator. An effective extraction was achieved by suspending a 1.0 µL
mixed drop of p-xylene and acetone (8:2 w/v) to the tip of a microsyringe immersed in a 2
mL donor sample solution and stirred at 400 rpm (Zhang et al., 2008). SDME technique
coupled with GC-NPD and GC-ECD has also been successfully applied for the
determination of multiclass pesticides in vegetable samples (tomato and courgette) by
Amvrazi & Tsiropoulos (2009). Donor sample solution preparation from solid vegetable
tissues was achieved in one step with the minimum amount of organic solvent (10% acetone
in water) and optimum SDME was accomplished using a toluene drop (1.6 µL) under mild
stirring for 25 min.
HS-SDME is very similar to direct SDME except that a microdrop of a high boiling
extracting solvent is exposed to the headspace of a sample. This technique allows rapid
stirring of the sample solution with no adverse impact on the stability of the droplet.
Additionally, as in headspace-solid phase microextraction (HS-SPME), non-volatile matrix
interferences are strongly reduced, if not totally eliminated. In this mode, the analytes are
distributed among three phases, the water sample, the headspace and the organic drop (Xu
et al., 2007). Aqueous phase mass transfer is the rate determining step in the extraction
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process as explained by Theis et al. (2001). Hence, a high stirring speed of the sample
solution facilitates mass transfer among the three phases. A HS-SDME was optimized for
the extraction of organochlorine and organophosphorous pesticide residues in food matrices
(cucumbers and strawberries) (Kin & Huat, 2009). The extraction was achieved by exposing
1.5 µL toluene drop to the headspace of a 5 mL aqueous solution in a 15 mL vial and stirred
at 800 rpm. The analytical parameters, such as linearity, precision, LOD, limits of
quantification (LOQ), and recovery, were compared with those obtained by HS-SPME and
solid-phase extraction. The mean recoveries for all three methods were all above 70% and
below 104%. HS-SPME was the best method with the lowest LOD and LOQ values. Overall,
the proposed HS-SDME- GC-ECD method was acceptable for the analysis of pesticide
residues in food matrices.
CFME was introduced by Liu & Lee, 2000, in order to improve the mass transfer between
aqueous and organic phases. The technique is based in the continually refreshing of the
surface of the immobilized organic drop used as extractant solvent by a constant flow of
sample solution delivered by an HPLC pumping system (Xu et al., 2007). Both diffusion and
molecular momentum resulting from mechanical forces contribute to its effectiveness. With
the use of an HPLC injection valve, precise control of the solvent drop size could be
achieved, avoiding the introduction of undesirable air bubbles. Another advantage was the
high enrichment factor that can be achieved, requiring smaller volumes of aqueous samples
for extraction (Xu et al., 2007). He & Lee (2006) reported the combination of CFME with
HPLC to extract and determine the widely-used organonitrogens and OPPs (simazine,
fensulfothion, etridiazole, mepronil and bensulide) (Table 1). CFME employs a single
organic solvent drop of carbon tetrachloride (3 µL) positioned at the tip of a polyether ether
ketone (PEEK) tubing, which is immersed in a continuous flowing aqueous sample solution
in a 0.5-mL glass chamber. The PEEK tubing acts as the organic drop holder and fluid
delivery duct. Analytes are partitioned between the organic drop and the bulk sample
solution. Important extraction parameters including type of solvent, volume, sample
solution flow rate, extraction time, pH and the addition of salts were investigated. Detection
limits lower than 4 µg/L were obtained for all analytes.
As mentioned above several parameters affect the rates and efficiencies of SDME techniques
such as: i) analyte properties, ii) solvent acceptor, iii) drop volume, iv) agitation, v) ionic
strength, vi) extraction time. A detailed discussion of these important parameters can be
found in the literature (Jeannota et al., 2010). i) Analyte properties: low molecular weight,
volatile and semi volatile analytes are extractable by headspace (HS-SDME). Direct
immersion (DI-SDME) extraction is appropriate for non polar or moderately polar high
molecular weight, semi volatile chemicals. Highly polar chemicals may need to be
derivatized to ensure recovery, especially when the matrix is aqueous. ii) Extractant solvent:
the extractant solvent in SDME is usually a pure or mixed hydrophobic solvent (n-hexane,
benzene, toluene, dichloromethane, n-butanol, etc.), although some authors have reported
the use of a hydrophilic solvent mixture as extractant solvent (p-xylene:acetone). iii) Drop
volume: the use of a large drop results in an increase of analyte extracted. However, larger
drops (>3 µL) are difficult to manipulate and less reliable. Difficulties with drop size
variations are minimized if the drop size used is about 1 µL. iv) Ionic strength: addition of
salts (such as NaCl or Na2SO4) to the sample may improve the extraction of analytes since
high ionic strength reduces their water solubility. However, apart from the salting-out
effect, the presence of salt can change the physical properties of the extraction film, thus
reducing the diffusion rates of the analytes into the drop. v) Agitation of the sample: the
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time required to thermodynamic equilibrium can be reduced by agitation. Three sample

agitation methods are available: stirring, vibration and vortexing. Stirring, using a magnetic
stir bar, is effective with stirring rates of 300–600 rpm for DI-SDME and 500–1000 rpm for
HS-SDME. The limitations of higher stirring rates are the dislodgement of the drop by the
sample solution or splashing when using headspace. Vibration and vortex stirring, used
with some autosamplers, are also effective, with the limitation that the agitation cannot
occur while the drop is exposed at the needle tip. vi) Extraction time: extraction efficiency
increases with longer extraction times in most of SDME techniques. The extraction time
should be enough to extract an adequate amount of analyte by the microdrops. Times
between 5 and 45 min are commonly used, longer times may cause drop dissolution.
Despite its simplicity, easy implementation, and low cost, SDME techniques have some
limitations, for example: i) direct immersion requires careful and intricate manual operation
because of problems of drop dislodgment and instability; ii) complex matrices requires a
pretreatment or extra filtration step; iii) sensitivity and precision of SDME methods even
acceptable need further improvement. The main issue lies with the adverse consequences of
prolonged extraction time and fast stirring rate, since they may result in drop dissolution
and/or dislodgement; and iv) SDME is not yet suitable as routinely applicable online
preconcentration procedure (Xu et al., 2007).
2.1.3 Hollow-fiber liquid-phase microextraction (HP-LPME)

Pedersen-Bjergaard & Rasmussen introduced hollow-fiber based liquid-phase
microextraction (HP-LPME) in 1999, to improve the stability and reliability of SDME
techniques (Pedersen-Bjergaard & Rasmussen, 1999). In HP-LPME the extracting phase was
placed inside the lumen of a porous polypropylene hollow fiber. The fiber had a porosity of
70% with a pore size of 0.2 µm, a wall thickness of 200 µm and an internal diameter of 600
µm. A supported liquid membrane was formed by dipping the hollow fiber into a suitable
organic solvent. The solvent penetrates the pores of the hollow fiber and bound by capillary
forces to the polypropylene network comprising the fiber wall. The high porosity enabled
immobilization of a considerable volume of solvent as a thin film, e.g. a 1 cm length of the
fiber was able to immobilize ca. 8 µL of solvent as a 200 µm film within the polypropylene
network. The extracting phase (acceptor solution) which was placed into the lumen of the
fiber was mechanically protected inside the hollow fiber and it was separated from the
sample by the supported liquid membrane (organic solvent), thus preventing its dissolution
into the aqueous sample. In LPME (HP-LPME), analytes are extracted from an aqueous
sample, through the organic solvent immobilized as supported liquid membrane (SLM),
into the acceptor solution placed inside the lumen of the hollow fiber. Subsequently, the
acceptor solution is removed by a micro-syringe and further analyzed (Pedersen-Bjergaard
& Rasmussen, 2008). Chemical principles of HP-LPME are similar to those employed in
supported liquid membrane (SLM), but the techniques differ in terms of instrumentation
and operation.
According to the analyte to be extracted, HP-LPME can be performed either in two-phase or
three-phase modes. In the two-phase LPME sampling mode, analyte is extracted from an
aqueous sample (donor phase) through a water-immiscible solvent immobilized in the pores
of the hollow fiber into the organic solvent (acceptor phase) present inside the hollow fiber
(Figure 2). In the three-phase LPME sampling mode, analyte is extracted from an aqueous
solution (donor phase) through the organic solvent immobilized in the pores of the hollow
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Table 1. Applications of SDME in the extraction of pesticide residues.
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Volume of
Extractant Stiring Extraction
Analytes Sample organic Detector LOD/LOQ
solvent speed (rpm) time (min)
solvent (µL)
Organochlorine (1) LOD: 0.0009-0.
Water (river) and
Triazole (1) Toluene 1.6 800 30 GC-ECD µg/L
wine (red wine)
Azole (2) LOQ: n.r.
Organophosphosphate LOD: 0.98-2.20
Juice (orange) Toluene 1.6 400 15 GC-FPD
(7) LOQ: n.r.
Water (lake) and
Organophosphosphate LOD: 0.21-0.56
juices (apple, Toluene 1.5 600 20 GC-FPD
(6) LOQ: n.r.
orange and pear)
Organophosphate (13) Water (farm, river Carbon LOD: 0.002-0.020
0.9 1300 40 GC-FPD
and well) tetrachloride LOQ: n.r.
Triazine (1)
Organophosphate (2) Carbon HPLC- LOD: 0.6-4.0 µ
Water 3.0 n.a. 10
Thiadiazole (1) tetrachloride UV LOQ: n.r.
Benzanilide (1)
Vegetable (cabbage, Acetone:p
LOD: 0.05-0.20
Organochlorine (9) cauliflower, xylene 1.0 400 30 GC-MS
LOQ: n.r.
Chinese cabbage) (2:8v/v)
Organophosphate (9)
Anilinopyrimidine (1)
Dicarboximide (1) GC-
Vegetables LOD: 0.03-30 µ
Triadiazine (1) Toluene 1.6 350 25 NPD/EC
(courgette, tomato) LOQ: n.r.
Strobilurin (1) D
Juvenile hormone
mimic (1)
Water and LOD: 0.022-0.101
Organochlorine (18) Toluene 2.0 380 37 GC-MS
wastewater LOQ: 0.074-0.337
EF, enrichment factor; n.r., not reported; n.a., no adjustment

fiber (organic phase) into another aqueous phase (acceptor phase) present inside the lumen
of the hollow fiber (Figure 2). The organic phase serves in this case as a barrier between the
acceptor and the donor aqueous solutions, preventing mixing of these two phases. Whereas
two-phase mode has been mainly used for hydrophobic compounds, further analyzed by
GC, three-phase mode has been preferably used for ionisable compounds, using LC or
capillary electrophoresis (CE) as analytical techniques (Psillakis & Kalogerakis, 2003).
Fig. 2. Schematic illustration of 2- and 3-phase LPME (from Pedersen-Bjergaard &

Rasmussen, 2008).
HP-LPME even providing high enrichment, an easy cleanup, low solvent consumption and
making possible the direct analysis by chromatography of the acceptor phase requires long
extraction times, which is perhaps the major disadvantage of the technique. Normally,
extraction time range between 15 and 45 min for sample volumes below 2 mL, whereas for 1
L samples even 2 h may be required to reach equilibrium (Pedersen-Bjergaard &
Rasmussen, 2008).
Recently, some proposals have been made in order to speed up the throughput of the
procedure, either by treating many samples in parallel, carrying out the extraction under
non-equilibrium condition (Ho et al. 2002), or using the so called dynamic hollow fiber
protected liquid phase microextraction (DHFP-LPME). The latter technique was successful
applied by Huang & Huang (2006) in the extraction of OCPs from green tea leaves and
ready-to-drink tea prior to GC–ECD analysis. In this work, six OCPs (heptachlor, aldrin,
endosulfan, p,p’-DDE, dieldrin and o,p’-DDT) were extracted and concentrated to a volume
of 3 µL of organic extracting solvent (1-octanol) confined within a 1.5 cm length of hollow
fiber. The effects of extractant solvent, extraction time and temperature, sample agitation,
plunger speed, and salt concentration on the extraction performance were investigated.
Good enrichments were achieved (34–297 fold) with this method, and good repeatabilities of
extraction were obtained, with RSDs below 12.57%. Detection limits were below 1 µg/L for
ready-to-drink tea and below 1 µg/g for green tea leaves. The application of HP-LPME to a
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large number of pesticides representatives of several chemical classes was reported by

Bolaños et al. 2008. In this study 50 pesticides were extracted from alcoholic beverages (wine
and beer) to a volume of 5 µl of organic extracting solvent (1-octanol) confined within a 2 cm
length of hollow fiber followed by ultra-high pressure liquid chromatography coupled to
tandem mass spectrometry (UHPLC–MS/MS), without any further clean-up step. Using
optimized conditions, low detection limits (0.01–5.6 µg/L) and acceptable linearity (R2 >
0.95) were obtained. Recently, a liquid-phase microextraction (LPME) based on
polypropylene hollow fiber was evaluated for the extraction of the fungicides
(thiabendazole, carbendazim and imazalil) from orange juices (Barahona et al., 2010). Each
sample aliquot (3 mL) was previously alkalinized with NaOH until reach a pH of 10-11, and
the analytes were further extracted through a supported liquid membrane (SLM) of 2-
octanone into 20 µL of a stagnant aqueous solution of 10 mM HCl inside the lumen of the
hollow fibre. Subsequently, the acceptor solution was directly subjected to analysis by LC-
MS and capillary electrophoresis (CE). The LC-MS provided better sensitivity than CE
allowing a LODs below 0.1 µg/L.
As described in the works above mentioned several parameters should be optimized in
order to obtain the maximum efficiency such as i) fiber, ii) organic solvent, iii) extraction
time, iv) temperature, v) agitation, vi) ionic strength and vii) pH (Psillakis & Kalogerakis,
2003). i) Fiber: the fiber should be hydrophobic and compatible with the organic solvents
used. Such requirements are met by fibers based on polypropylene; most of them have 600
mm of inner diameter, compatible with the volumes (µL) of the acceptor solution required
for microextraction. ii) Organic solvent: a fundamental step in the optimization of the LPME
methods is the selection of the organic solvent. Some properties need to be considered in
their choice including: water-immiscibility, to prevent the organic phase dissolution in the
aqueous (donor) phase; low volatility, to avoid organic phase loss during extraction;
compatibility with the fiber used; easy immobilization within the pores of the hollow fiber;
and high solubility for target analytes. iii) Extraction time: mass-transfer is a time-dependent
process, increasing with the time of extraction. In practice to ensure high sample
throughputs sampling times are shorter than the total chromatographic run time. iv)
Agitation: agitation of the sample is routinely applied to accelerate the extraction kinetics.
Increasing the agitation rate of the donor solution enhances extraction, the diffusion of
analytes through the interfacial layer of the hollow fiber is facilitated, and the repeatability
of the extraction method is improved. v) Temperature: with increasing temperature, the
diffusion coefficients also increase in response to decreased viscosity. Thus the time
required to reach equilibrium decrease. On the other hand, partition coefficients for the
acceptor phase decrease, reducing the amount of analyte extracted. Therefore, the speed of
extraction could be improved at costs of a loss of sensitivity. Typically, LPME is performed
at room temperature in order to avoid possible bubbles problems and evaporation of the
solvent during extraction, since the amount of solvent used is very small (20 µL). vi) Ionic
strength: depending on the nature of the target analytes, addition of salt to the sample
solution can decrease their solubility and therefore enhance extraction because of the
salting-out effect, in particular for polar analytes. Among the salts mainly used sodium
chloride is the most common. vii) pH: sample pH is crucial for efficient extraction of acidic
and basic analytes. pH adjusting results in a greater ratio of distribution, ensures high
enrichment factors and high recovery of the analytes of interest. Adjustments in pH can
increase the extraction efficiency, since both the balance dissociation and the solubility of
acids and bases are directly affected by sample pH.
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HP-LPME provides in general an acceptable sensitivity in the analysis of pesticide residues.

However, extraction procedure requires the presence of the analytes in liquid solutions,
being its application usually restricted to liquid samples. Moreover the technique is difficult
or even impossible to automate, the time of extraction could be considered too long and the
operator skills should be high in order to get reproducible results.
2.1.4 Dispersive liquid-liquid microextraction (DLLME)

Dispersive liquid-liquid microextraction (DLLME) was developed by Assai and co-workers
in 2006 (Rezaee et al., 2006). Consists in the rapid addition to an aqueous sample (in a
conical test tube) of a mixture of two selected solvents (few microliters of a water-immiscible
high density extractant solvent jointly with a dispersive solvent with high miscibility in both
extractant and water phases). The aim is to form a cloudy solution of small droplets of
extractant solvent which are dispersed throughout the aqueous phase. In consequence of the
very large surface area formed between the two phases, hydrophobic solutes are rapidly
and efficiently enriched in the extractant solvent and, after centrifugation, they can be
determined in the phase settled at the bottom of the tube. The resultant sedimented phase is
read for direct analysis by GC or LC.
Since its introduction, DLLME has gained popularity as a simple, fast and reliable tool for
sampling preparation of a variety of analytes, as can be seen in recent reviews (Xiao-Huan et
al., 2009; Ojeda & Rojas, 2009; Rezaee et al. 2010; Herrera-Herrera et al., 2010). DLLME has
extensively been used for direct extraction of pesticides from aqueous samples such water,
fruit juice and wine (Table 2). The first study using DLLME in pesticide residues was
applied in the extraction of 13 OPPs (phorate, diazinon, disolfotane, methyl parathion,
sumithion, chloropyrifos, malathion, fenthion, profenphose, ethion, phosalone, azinphose-
methyl, co-ral) from river water (Berijani et al., 2006). In this study a mixture of 12.0 L of
chlorobenzene (extractant solvent) and 1.00 mL of acetone (dispersive solvent) was rapidly
injected in 5 mL of aqueous sample. The sedimented phase (about 5 µL) collected after
centrifugation (2 min at 5000 rpm) was analyzed by GC-FPD. Some important parameters,
such as kind of extractant and dispersive solvents and their volumes, extraction time,
temperature and salt effect were investigated. Under the optimized conditions, enrichment
factors and extraction recoveries were high, ranging between 789–1070 and 78.9–107%,
respectively. LODs ranged between 3 and 20 pg/mL for most of the analytes. Other classes
of pesticides were extracted by DLLME from water such as triazine herbicides, amide
herbicides, phenylurea herbicides, organochlorines, pyretroids and carbamates (Table 2). In
most of the reported studies only one chemical class of pesticides was evaluated, being the
number of pesticide residues scarce (less than eighteen analytes). However, in a recent
publication different classes of pesticides namely triazole fungicides, isoxazolidinone
herbicides and carbamates were simultaneously evaluated, although the number of analytes
pertaining at each class has been reduced (three) (Caldas et al., 2010). After optimization of
the parameters that influence the extraction efficiency, such as the type and volume of the
dispersive and extractant solvents, extraction time, speed of centrifugation, pH and addition
of salt, the extraction of pesticide residues from 5 mL of water was achieved with a mixture
of 2.0 mL acetonitrile (dispersive solvent) containing 60 µL of carbon tetrachloride
(extractant solvent), followed by centrifugation at 2000 rpm for 5 min; the analysis was
performed by LC-MS/MS. The recoveries of pesticides in water at spiking levels between
0.02 and 2.0 µg/L ranged from 62.7% to 120.0%. RSDs varied between 1.9% and 9.1%. LOQs
of the method considering a 50-fold preconcentration step were 0.02 µg/L. The LODs of the
method were not reported in this study.
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The application of the DLLME procedure in the extraction of pesticide residues in food
samples is reported in only few papers, probably due to the complexity of food matrices
(Table 2). Montes et al. (2009) used DLLME for preconcentration of seven fungicides
(metalaxyl-M, penconazole, folpet, diniconazole, propiconazole, difenoconazole and
azoxystrobin) in wine samples after extraction with SPE. A direct use of DLLME as
extraction procedure followed by GC-MS analysis was performed by Cunha et al. (2009) to
determine 24 pesticide residues, belonging at eight different chemical classes, in juice fruits.
In order to avoid the precipitation of some components of the matrix, which make
unsuitable the application of DLLME as referred by Montes et al (2009), samples were
centrifuged prior extraction. As can be seen in Figure 3, the optimized DLLME procedure
5 mL of Add 400 µL of Centrifuge 2 min Remove the sedimented phase (85 µl)
sample acetone with 100 µL at 2000 rpm and transfer into an autosampler vial
of provided with an insert
carbon tetrachloride
Fig. 3. Diagram of the dispersive liquid–liquid microextraction procedure used by Cunha et
al. (2009).
consisted in the formation of a cloudy solution promoted by the fast addition to the sample
(5 mL) of a mixture of carbon tetrachloride (extractant solvent, 100 µL) and acetone
(dispersive solvent, 400 µL). The tiny droplets formed and dispersed were sedimented (85
µL) in the bottom of the conical test tube after centrifugation at 2000 rpm for 2 min. More
than the parameters that influence the extraction efficiency of DLLME such as type and
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volume of extractant solvent, type and volume of dispersive solvent and salt addition, other
factors that could restrict the analytical performance, such as matrix effects or robustness of
the method were evaluated according the Sanco guidelines (2007). Under the optimized
conditions mean recoveries for apple juice spiked at three concentration levels ranged from
60% to 105% and the intra-repeatability ranged from 1% to 21%. The LODs of the 24
pesticides ranged from 0.06 to 2.20 µg/L. In 2 of a total of 28 analysed fruit juice samples
residues of captan were found, although at levels below the maximum legal limit
established by European Union (Figure 4).
DLLME is more suitable for the extraction of analytes from aqueous samples; nonetheless,
some authors have applied this process in solid samples after an adequate pretreatment.
Zhao et al. (2007) applied DLLME as a concentration procedure after a previous extraction
with QuEChERS of OPPs (ethoprophos, parathion methyl, fenitrothion, malathion,
chlorpyrifos and profenofos) from watermelon and cucumber. Hence, 1 mL of the extract
obtained after homogenization of 10 g of sample with 10 mL of acetonitrile, 4 g MgSO4, and
1 g NaCl, was added with 27 µL of chlorobenzene and rapidly injected in 5 mL of water.
Then 1 µL of 18 µL of sedimented phase obtained by centrifugation of the mixture at 4000
rpm for 3 min was analyzed by GC-FPD. The optimized method allowed recoveries between
67 and 111%, repeatability between 2 and 9% and LODs ranging from 0.010 to 0.190 µg/kg,
for all the target pesticides. In other study, Zang et al. (2008) applied the DLLME procedure
directly in the extraction of captan, folpet and captafol from apples. The developed
procedure consisted in the injection of a mixture containing chlorobenzene (extractive), and
acetone (dispersive) directly into an aqueous extract of apple samples, obtained after
homogenization with a solution of zinc acetate dehydrate and dilution with water. Under
the optimum conditions, high enrichment factors for the targets were achieved ranging from
824 to 912. The recoveries of fungicides in apples ranged from 93.0 to 109.5% and the RSD
ranged from 3.8 to 4.9%. The LODs were between 3.0 and 8.0 g/kg.
To date, the majority of the applications related to DLLME involve the use of solvents of
high, density commonly chlorinated solvents (e.g. chlorobenzene, carbon tetrachloride and
tetrachloroethylene) as extractant solvents. However, the use of ionic liquids (IL) as
extractants has been found to be especially important in DLLME as well as in other
microextraction procedures (in order to replace the volatile ones used during sample
preparation procedures) because of their negligible vapor pressure, good solubility for
organic and inorganic compounds, no flammability, high thermal stability, wide
temperature range as a liquid phase, etc. (Han & Armstrong, 2007; Ravelo-Pérez et al., 2009).
One of the main drawback of the use of IL in DLLME is the impossibility to make use of GC
in the analysis, due to the adverse effects of these solvents in the chromatographic system.
IL-DLLME has been applied in the extraction of a high variety of pesticides in water and
food matrices such as fruits and honey, as can be seen in Table 2. DLLME based on IL was
initially applied by Zhou et al. (2008a), to extract five pyrethroid pesticides (cyhalothrin,
deltamethrin, fenvalerate, taufluvalinate and biphenthrin) in different types of water
samples (tap, river and reservoir water, and groundwater). In this study, the sample (10 mL)
was heated at 80 ºC after addition of 45 µL of 1-hexyl-3 methylimidazolium
hexafluorophosphate [C6MIM][PF6]. The IL mixed with the solution entirely at this
temperature and thereafter the solution was cooled with ice-water for a certain time. The IL
and the aqueous phase were separated after centrifugation and the IL phase injected into the
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A)
B)
Fig. 4.A) Chromatogram of spiked blank apple juice with 24 OPPs B) Overlay of extracted
ion chromatograms in SIM mode for captan (ion 149) obtained in not contaminated (- - -)
and contaminated (___) (0.541 µg/L) apple juice samples using DLLEM extraction and
MDGC-MS analysis (from Cunha et al., 2009).
HPLC-UV. In this study good recoveries were obtained (76.7– 135.6%) and LODs were in
the range 0.28–0.6 g/L. In a further work, the same group used a similar procedure, using
[C6MIM][PF6] as extractant solvent in DLLME at 80ºC for determine traces of
methylparathion and phoxim in water (Zhou et al., 2008b). A new IL-DLLME procedure
was introduced by Liu et al. (2009) for the extraction of four insecticides (fipronil,
chlorfenapyr, buprofezin, and hexythiazox) from water. The proposed procedure combined
extraction and concentration of the analytes into one step, avoiding heating and cooling
steps, so reducing extraction time. Thus, a mixture of 0.052 g [C6MIM][PF6] and 0.50 mL
methanol (dispersive solvent) was quickly injected into the sample (5.0 mL). Then, the
mixture was centrifugated at 4000 rpm for 10.0 min, and 19 µL of sedimented phase were
diluted with 50 µL methanol and 10 µL of the misture analysed by HPLC-UV. Under the
optimized conditions, good enrichment factors (209–276) and accepted recoveries (79–110%)
were obtained for the extraction of the target analytes in water samples. The LODs for the
four insecticides ranged from 0.53 to 1.28 µg/L.
The application of IL-DLLME to solid samples is scarce as referred above for the classical
DLLME. Usually, it is necessary a previous pretreatment of the sample in order to obtain an
aqueous extract before extraction. In a recent work Wang et al. (2010) developed an IL-
DLLME/HPLC-UV method for the extraction and determination of triazines in honey. A
mixture of 175 µL of [C6MIM][PF6] (extractant solvent) and 50 µL of 10% Triton X 114
(dispersive solvent) was rapidly injected into 20 mL aqueous honey sample, obtained by
dissolution of 2 g of honey with 20 mL of water. The detection limits for chlortoluron,
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prometon, propazine, linuron and prebane were 6.92, 5.84, 8.55, 8.59 and 5.31 µg/kg,
respectively.
Another type of extractant solvents used in DLLME are low density solvents such as
undecanol, 1-dodecanol, 2-dodecanol and n-hexadecane, which are usually less toxic than
the chlorinated solvents. An interesting work was developed by Leong & Huang (2009) for
the determination of OCPs in water samples. The method is based on the solidification of a
floating organic drop (DLLME-SFO) and it is combined with GC-ECD. The dispersive
solvent (200 µL of acetonitrile) containing 10 µL of hexadecane (HEX) was rapidly injected
into 5.0 mL water sample. After centrifugation, the fine HEX droplets (6±0.5 µL) floating at
the top of the screw-capped tube were solidified through ice and then transferred into a vial
to be injected into GC. Under optimum conditions, enrichment factors and extraction
recoveries are high ranging between 37–872 and 82.9–102.5%, respectively. LODs ranged
between 0.011 and 0.110 µg/L for most of the analytes. Recently Chen et al. (2010) reported a
low-density extractant solvent-based, termed solvent terminated (ST) DLLME to determine
carbamate pesticides (carbofuran, tsumacide, isoprocarb, and pirimicarb) in water by GC-
MS/MS. Hence, 0.50 mL of acetonitrile containing 15 L of toluene were rapidly injected in
5 mL of water. After dispersing, the obtained emulsion was quickly cleared into two phases
when an aliquot of acetonitrile (0.5 mL) was introduced as a chemical demulsifier into the
aqueous bulk. Therefore, the developed procedure does not need centrifugation to achieve
phase separation. Under the optimized conditions, the LODs for all the target carbamate
pesticides were in the range of 0.001–0.50 µg/L and the precisions were in the range of 2.3–
6.8%.
In order to achieve such a wide range of applications, several parameters have to be taken
into account to optimize DLLME to extract pesticide residues, such as i) type and volume of
extractant solvent, ii) type and volume of dispersive solvent, iii) extraction time, and iv)
effect of salt addition. i) Extractant solvent: the extractant solvents should be immiscible
with water, and they must possess both good solubility for analytes and good
chromatographic behavior. They can either have higher or lower density than water and the
volume used ranged between 10 to 100 µL. Lower volumes of extractant solvent enhance
enrichment factor, although reducing the volume of sedimented phase, could give problems
of reproducibility. ii) Dispersive solvent: the dispersive solvent should be miscible with both
aqueous sample and extractant solvent and possess the capacity to decrease the interfacial
tension of extractant solvent in order to make the droplet size smaller, increasing the
extraction efficiency. Acetone, methanol and acetonitrile can be used as dispersive solvents
at volumes ranging from 0.5 mL to 2 mL. iii) Extraction time: in DLLME after mixture of the
three components (sample, extractant and dispersive solvent) the equilibrium is achieved in
few seconds due to the large contact surface between tiny drops of extractant solvent and
the sample. Nevertheless, in most of the studies the extraction time ranged from 1 to 5 min.
iv) Salt addition: salt addition can improve extraction yield in DLLME, particularly for those
analytes with lower solubility, as a result of a “salting out” effect. This effect is prevailing in
DLLME when NaCl is employed.
DLLME has generally showed a very good performance to extract pesticide residues from
water and aqueous extracts of food samples, but it is desirable to extend this application to
more complex matrices and to a large number of pesticide residues using standard
guidelines for the validation of the methods.
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Table 2. Applications of DLLME in the extraction of pesticide residues
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Dispersive Re
Analytes Sample Extractant solvent Detector LOD/LOQ
solvent
Water (river Chlorobenzene Acetone LOD: 0.003-0.020 µg/L
Organophosphate (13) GC-FPD 9
and farm) (12 µL) (1.0 mL) LOQ: n.r.
Watermelon Chlorobenzene Acetonitrile
Organophosphate (6) LOD: 0.5-20 µg/kg
and (27 µL) (1.0 mL) GC-FPD 6
LOQ: n.r.
cucumber
Water (river Tetrachloroethane Methanol LOD: 1.0 µg/L
N-methylcarbamate (1) HPLC-UV 9
and lake) (20 µL) (0.5 mL) LOQ: n.r.
Organophosphate (1)
Chlorobenzene Acetone LOD: 3.0-8.0 µg/kg
Phathalimide (1) Apples GC-ECD 9
(9 µL) (1.0 mL) LOQ: n.r.
Carboximide (1)
Acetone
Water (river, Chlorobenzene LOD: 0.04-0.10 µg/L
Pyrethroid (3) (1 mL) GC-ECD 7
well and tap) (15 µL) LOQ: n.r.
Water (tap,
Pyrethroid (5) [C6MIM][PF6] LOD: 0.28-0.6 µg/L
river, reservoir n.a. HLPC-UV 7
(45 µL) LOQ: n.r.
and ground)
Water (rain,
Organophosphate (2) [C6MIM][PF6] LOD: 0.17-0.29 µg/L
river, reservoir n.a. HLPC-UV 8
(50 µL) LOQ: n.r.
and ground)
Water (tap,
lake, river, well
Organophosphate (5) Carbon tetrachloride Methanol
and farm) LOD: 0.21-3.05 µg/L
Carbamate (1) (10 µL) (0.8 mL) GC-FPD 7
green tea and LOQ: n.r.
tea
leaves
Table 2. Applications of DLLME in extraction of pesticide residue (cont.)
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Analytes Sample Extractant solvent Dispersive Detector LOD/LOQ Rec

solvent
Organophosphate (13) Juice (apple) Carbon tetrachloride Acetone GC-MS LOD: 0.06-2.20 µg/L 60
Pyrethroid (3) (100 µL) (0.4 mL) LOQ: 0.2-7.3 µg/L
Phathalimide (1)
Dicarboximide (1)
Phenylamide (1)
Cyclodiene (1)
Anilino-pyrimidine (1)
Strobilurin (1)
Carbamate (5) Water (surface) Trichloromethane Acetonitrile HPLC-DAD LOD: 0.1-0.4 µg/L 8
(40 µL) (1 mL) LOQ: n.r.
Phenyurea (8) Water (river, tap Carbon disul de Acetone HPLC- LOD: 0.01-0.5 µg/L 86
and well) (103 µL) and toluene (2 mL) UV/DAD LOQ: n.r.
(45 µL)
Carbamate (1) Water (river and Tetrachloroethane Acetonitrile HPLC-FLD LOD: 0.0123-0.016 µg/L 80
Organophosphate (1) tap) and juice (15 µL) (1.0 mL) LOQ: n.r.
(apple, peach
and grape)
Organochlorine (5) Water (river, sea Tetrachloroethylene tert-butyl methyl GS-MS LOD: 0.0004-0.0025 µg/L 54
and reservoir) (5.2 µL) ether LOQ: n.r.
( 7.8 µL)
Pyrazole (1) Water (tap, lake [C6MIM][PF6] Methanol HPLC-DAD LOD: 0.53-1.28 µg/L 79
Thiazolidine (2) and fountain) (0.052 g) (0.5 mL) LOQ: n.r.
Pyrrole (1)
Organophosphate (4) Water (river, [C8MIM][PF6] Methanol HPLC-UV LOD: 0.1- 5.0 µg/L 87
tap, rain and (35 µL) (1.0 mL) LOQ: n.r.
well)
Table 2. Applications of DLLME in extraction of pesticide residue (cont.)
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Analytes Sample Extractant solvent Dispersive Detector LOD/LOQ Re

solvent
Benzimidazole (1) Bananas [C6MIM][PF6] Methanol HPLC-DAD LOD: 0.320-4.66 µg/kg 53
Carbamate (2) (88 mg) (0.714 mL) LOQ: n.r.
Dicarboximide (1)
Quinazoline (1)
Triazole (1)
Diphenyl ether (1)
Thiazolidine (1)
Organochlorine (18) Water (river, Tetrachloroethylene Acetone GC-MS LOD: 0.001-0.025 µg/L 5
surface and tap) (10 µL) (1.0 mL) LOQ: n.r.
and wastewater
Organophosphate (1) Wine (red and 1,1,1-trichloethane Acetone GS-MS LOD: n.r. 7
Strobilurin (1) white) (100 µL) (1 mL) LOQ: 0.020-.020 µg/L
Phenylamide (1)
Triazole (1)
Conazole (1)
Azole (1)
Organochlorine (6) Water (lake and Hexadecane Acetonitrile GC-ECD LOD: 0.011–0.109 µg/L 8
tap) (10 µL) (0.2 mL) LOQ: n.r.
Organophosphate (10) Tea N-hexane Acetonitrile GC-FPD LODs: 0.030-1.00 µg/kg 8
(24 µL) (0.5 mL) LOQ: n.r.
Triazole (2) Water (tap) Carbon tetrachloride Acetonitrile LC-MS/MS LOD: n.r. 6
Carbamate (1) (60 µL) (2.0 mL) LOQ: 0.02 µg/L
Carbamate (4) Water (lake) Toluene Acetonitrile GS-MS LOD: 0.001-0.050 µg/L 9
(50 µL) (1.0 mL) LOQ: n.r.
Triazine (5) Honey [C6MIM][PF6] (175 n.a HPLC-DAD LOD: 5.31–8.59 µg/kg 6
µL) and 10% Triton X LOQ: n.r.
114 (50 µL)
EF, enrichment factor; n.r., not reported; n.a. no adjustment
2.2. Analysis
The determination of pesticide residues in water and food matrices has traditionally been
performed by GC, due the high number of theoretical plates of the columns employed and
the variety and selectivity capabilities of the detectors than can be coupled such as ECD,
NPD, and FPD. Among the detectors used, MS is the preferred tool for determination of
multi class pesticide residues because it permits: i) the simultaneous quantification and
identification of detected analytes; ii) the detection of a wide range of analytes
independently of its elemental composition; iii) mass-spectrometric resolution of co-eluting
peaks; and iv) potentially faster analysis time (Cunha et al., 2010).
To increase sample throughput during GC analysis, which would consequently reduce the
laboratory operating costs, several approaches were evaluated such as the reduction of:
column length, column inner diameter or column stationary film thickness; and the
utilization of fast temperature programming, low-pressure and multicapillary columns
(Maštovská & Lehotay 2003). In practice a combination of two or more approaches is very
often applied to enhance the speeding-up effect with the less sacrifice in sample capacity
and/or separation efficiency. Sample capacity influences the limit of detection and the
sensitivity, for example. Separation efficiency influences performance characteristics such as
selectivity, detection limit (through the level of chemical noise) and, of course, accuracy of
the analytical results. Multidimensional GC system with Deans switch heart-cutting
represents a very interesting technical solution, which not only responds adequately to the
demand of increased speed of analysis, capacity and separation efficiency, but also provided
an enhancement in robustness. This technique is based essentially on the transfer of selected
effluent fractions from a first to a second column for MS analysis and transfer of fractions
without analytical interest to a restrictor column for waste (see Figure 5) (Cunha et al., 2009;
Cunha & Fernandes, 2010 ). A devoted transfer device (Deans switch), situated between the
two columns, enables the entire procedure.
Recently a dual GC column system involving a short wide-bore capillary column connected
by a Deans switch device to a narrower and longer second chromatographic column was
successful applied in determination of 24 pesticide residues in fruit juice (Cunha et al., 2009).
This system allowed a gain in the speed of chromatographic analysis, providing an efficient
sample injection and column introduction of the analytes with limited interferences, high
sample capacity, and sharp and symmetric peak shapes without loss of resolution.
Notwithstanding the recent advances in GC-MS systems, the analysis of polar, non-volatile
or/and thermally labile pesticides by this technique is limited, usually requiring chemical
derivatization. LC-MS/MS has become a standard approach in developed countries to
expand the range of pesticides quantified and identified in complex matrices.
3. Conclusions
Microextraction methods usually require both smaller sample size and organic solvent
volumes when compared with the conventional methods. The main advantages of these
procedures are the high degree of enrichment for the analytes in complex matrices, which
enable detection limits down to the levels required by the regulatory bodies to the analysis
of pesticide residues in water and food. Additionally, given the compatibility of the solvents
used, and the low volumes involved, the procedures are easily associated with gas or liquid
chromatography. Most of microextraction applications are employed in aqueous samples for
the extraction of nonpolar or moderately polar high molecular weight analytes. Although
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Fig. 5. Deans switch GC–MS system. (A) The solenoid valve is in the on position, allowing
effluent to flow to the 2D GC separation column prior to MS detection. (B) The solenoid
valve is in the off position and effluent from the primary column is flowing to the exit gas
line. (Adapted from Agilent).
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some attempts were made for the extraction of analytes in solid matrices and also for the
extraction of polar analytes, is still expected an increment along this line in the future. On
other hand, despite their high-throughput, the automation of most of microextraction
procedures presented seems to be very difficult and has not yet been achieved, thus new
developments in this area are required.
4. Acknowledgments
S.C.C. is grateful to “POPH-QREN- Tipologia 4.2, Fundo Social Europeu e Fundo Nacional
MCTES”. This research was supported by grant from the FCT project “PTDC/AGR-
ALI/101583/2008” and Compete/ FEDER.
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Pesticides - Strategies for Pesticides Analysis
Edited by Prof. Margarita Stoytcheva
ISBN 978-953-307-460-3
Hard cover, 404 pages
Publisher InTech
Published online 21, January, 2011
Published in print edition January, 2011
This book provides recent information on various analytical procedures and techniques, representing
strategies for reliability, specificity, selectivity and sensitivity improvements in pesticides analysis. The volume
covers three main topics: current trends in sample preparation, selective and sensitive chromatographic
detection and determination of pesticide residues in food and environmental samples, and the application of
biological (immunoassays-and biosensors-based) methods in pesticides analysis as an alternative to the
chromatographic methods for "in situ" and "on line" pesticides quantification. Intended as electronic edition,
providing immediate "open access" to its content, the book is easy to follow and will be of interest to
professionals involved in pesticides analysis.
How to reference
In order to correctly reference this scholarly work, feel free to copy and paste the following:
Sara C. Cunha, J.O. Fernandes and M. Beatriz P.P. Oliveira (2011). Current Trends in Liquid-Liquid
Microextraction for Analysis of Pesticide Residues in Food and Water, Pesticides - Strategies for Pesticides
Analysis, Prof. Margarita Stoytcheva (Ed.), ISBN: 978-953-307-460-3, InTech, Available from:
http://www.intechopen.com/books/pesticides-strategies-for-pesticides-analysis/current-trends-in-liquid-liquid-
microextraction-for-analysis-of-pesticide-residues-in-food-and-water
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1

Current Trends in Liquid-Liquid Microextraction

2. Analytical tools for determination of pesticide residues in food and water

2.1 Sample preparation

organic solvents (Paleologos et al., 2005). CPE or micelle-mediated extraction, is based on

2.1.2 Single drop microextraction (SDME)

Fig. 1. Schematic illustration of direct immersion single-drop microextraction (from Xu et al.,

time required to thermodynamic equilibrium can be reduced by agitation. Three sample

2.1.3 Hollow-fiber liquid-phase microextraction (HP-LPME)

EF, enrichment factor; n.r., not reported; n.a., no adjustment

Fig. 2. Schematic illustration of 2- and 3-phase LPME (from Pedersen-Bjergaard &

large number of pesticides representatives of several chemical classes was reported by

HP-LPME provides in general an acceptable sensitivity in the analysis of pesticide residues.

2.1.4 Dispersive liquid-liquid microextraction (DLLME)

Analytes Sample Extractant solvent Dispersive Detector LOD/LOQ Rec

Analytes Sample Extractant solvent Dispersive Detector LOD/LOQ Re

tandem mass spectrometry for the determination of carbamate pesticides in water

Saraji, M. & Tansazan, N. (2009). Application of dispersive liquid–liquid microextraction for

InTech Europe InTech China

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