Trends in Analytical Chemistry 71 (2015) 169–185

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Trends in Analytical Chemistry

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a c

Evolution and applications of the QuEChERS method

M.Á. González-Curbelo a,b, B. Socas-Rodríguez a, A.V. Herrera-Herrera a,
J. González-Sálamo a, J. Hernández-Borges a, M.Á. Rodríguez-Delgado a,*
a Departamento de Química, Unidad Departamental de Química Analítica, Facultad de Ciencias, Universidad de La Laguna (ULL), Avenida Astrofísico

Francisco Sánchez, s/n°. 38206, San Cristóbal de La Laguna, Tenerife, España

b
Departamento de Ciencias Básicas, Universidad EAN, Calle 74 n° 9-49, Bogotá, Colombia

A R T I C L E I N F O A B S T R A C T

Keywords:
It is widely recognized that the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method is rel-
Bioanalysis
evant in pesticide residue analysis. Many official laboratories around the globe are routinely using it due
Environment
Extraction
to the advantages encapsulated in its name. However, the frontiers of the application of QuEChERS are
Food not yet established. The method is effective for the analysis of other groups of compounds, including phar-
Mycotoxin maceuticals, mycotoxins, and polycyclic aromatic hydrocarbons, in a wide variety of complex matrices.
Pesticide This review article provides a general overview of the most relevant modifications of the QuEChERS method
Pharmaceutical that have been widely accepted and applied (including both extraction and clean-up) and a general view
Polycyclic aromatic hydrocarbon of the different groups of compounds to which it has been fruitfully applied. We do not include those
QuEChERS approaches where only half the method has been used.
Sample pretreatment
© 2015 Elsevier B.V. All rights reserved.

Contents

1. Introduction ........................................................................................................................................................................................................................................................ 170

2. The original QuEChERS method ................................................................................................................................................................................................................... 170
3. Most relevant modifications ......................................................................................................................................................................................................................... 171
4. Application fields .............................................................................................................................................................................................................................................. 172
4.1. Pesticide analysis ................................................................................................................................................................................................................................. 174
4.2. Pharmaceutical analysis .................................................................................................................................................................................................................... 174
4.3. Mycotoxin analysis ............................................................................................................................................................................................................................. 175
4.4. PAH analysis .......................................................................................................................................................................................................................................... 178
4.5. Miscellaneous ....................................................................................................................................................................................................................................... 180
5. Conclusions ......................................................................................................................................................................................................................................................... 182
Acknowledgements .......................................................................................................................................................................................................................................... 182
References ............................................................................................................................................................................................................................................................ 182

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; ACN, Acetonitrile; Al-N, Alumina neutral; AMPA, Aminomethylphosphonic acid; BDMC, 4-bromo-3,5-dimethylphenyl-
N-methylcarbamate; BPA, Bisphenol A; BPB, Bisphenol B; BSA, Bis-(trimethylsilyl)acetamide; C18, Octadecylsilane; CCα, Decision limit; CE, Capillary electrophoresis; DART,
Direct analysis in real time; di-Na, Sodium citrate dibasic sesquihydrate; DLLME, Dispersive liquid-liquid microextraction; DON, Deoxynivalenol; d-SPE, Dispersive solid-
phase extraction; EtOAc, Ethyl acetate; EPA, Environmental Protection Agency; EU, European Union; FD, Fluorescence detection; FI, Flow injection; FIA, Flow-injection analysis;
GC, Gas chromatography; GCB, Graphitized carbon black; HMF, 5-hydroxymethylfurfural; HOAc, Acetic acid; HRMS, High-resolution mass spectrometry; IAC, Immunoaffinity
chromatography; IS, Internal Standard; LC, Liquid chromatography; LCL, Lowest calibration level; LIF, Laser-induced fluorescence; LLE, Liquid-liquid extraction; LPGC, Low-
pressure gas chromatography; MBZ, Mebendazole; MRL, Maximum Residue Limit; MS, Mass spectrometry; MSPD, Matrix solid-phase dispersion; MWCNT, Multi-walled
carbon nanotube; NaOAc, Sodium acetate; OCP, Organochlorine pesticide; PAH, Polycyclic aromatic hydrocarbon; PBDE, Polybrominated diphenyl ether; PCB, Polychlori-
nated biphenyl; PSA, Primary-secondary amine; Q, Quadrupole; QqQ, Triple quadrupole; SAX, Strong anion exchange; SMX-d4, Sulfamethoxazole-d4; SPE, Solid-phase extraction;
TMCS, Trimethylchlorosilane; TMSI, N-trimethylsilylimidazole; TPM, Triphenylmethane; TPP, Triphenyl phosphate; tri-Na, Sodium citrate tribasic dehydrate; TSL, Terbium-
sensitized luminescence; UHPLC, Ultra-high-performance liquid chromatography; ZEN, Zearalenone; Z-Sep, Zirconium-oxide base sorbent.
* Corresponding author. Tel.: +34 922 31 80 46; Fax: +34 922 31 80 05.
E-mail address: mrguez@ull.edu.es (M.Á. Rodríguez-Delgado).

http://dx.doi.org/10.1016/j.trac.2015.04.012
0165-9936/© 2015 Elsevier B.V. All rights reserved.
170 M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185
1. Introduction
In 2003, when Michelangelo Anastassiades (visiting scientist from
Chemisches und Veterinäruntersuchungsamt, Stuttgart, Germany)
and Steven J. Lehotay (US Department of Agriculture, Philadel-
phia, Pennsylvania, USA) published along with their co-workers their
paper, Fast and easy multi-residue method employing acetonitrile
extraction/partitioning and “dispersive solid-phase extraction” for
the determination of pesticide residues in produce [1], probably none
of the inventors could imagine the relevance that their developed
quick, easy, cheap, effective, rugged and safe (QuEChERS) method
would achieve in the scientific community.
When any scientist proposes a new method, we always hope to
come up with an interesting and useful contribution to the field,
but the QuEChERS method is indeed a major development.
For some scientists, the procedure introduced in 2003 was not
completely new. Obviously, solid-liquid extraction/partitioning (the
first step) was well known (also including the salting out) and applied
to the extraction of a wide variety of analytes and matrices. Re-
garding the d-SPE step (not named in that way at that time), sorbents
were also already applied for sample clean-up though mostly in SPE
cartridges. However, the unique combination of procedures (ex-
traction and clean-up), solvents, salts and sorbents was found to
be very effective, up to the point that it is currently the standard
sample-preparation procedure for pesticides analysis in fruits and
vegetables with further extensions to other applications.
As a result of the inherent advantages of the method, with “Green
Chemistry” characteristics [2], the method quickly expanded to the
extraction of different groups of compounds from environmental,
agroalimentary and bioanalytical matrices. Its great flexibility has
shown that many modifications (including sample sizes) can still
provide high recoveries; consequently, there are many more
QuEChERS versions than desirable. A good number of vendors cur-
rently sell QuEChERS kits to make the procedure faster, to avoid
weighing the salts, and adapted to each need. Furthermore, despite
the method being very quick, efforts were recently made to fully
automate the sample-preparation procedure [3–7].
Clearly, any review article devoted to the QuEChERS method faces
two important problems:
• there are many manuscripts that have used the method; and,
• many of them claim to have used it, when only the partition-
ing step or the d-SPE procedure alone has been used [8,9].
That is why we aim to provide a general overview of the evo-
lution of the full method, that is, the main changes or modifications
that have provided relevant results in sample preparation. Further-
more, we place special emphasis on showing the wide variety of
matrices and analytes that have been successfully extracted with
this method.
2. The original QuEChERS method
In the original manuscript, the QuEChERS method was devel-
oped for the extraction of multi-class pesticide residues from fruits
and vegetables [1]. GC-MS analyses were carried out employing TPP
as IS. After suitable homogenization of the samples, they were first
solvent extracted/partitioned and then the extract was cleaned up
by d-SPE with the aim of eliminating the possible interfering com-
pounds from the food extracts (e.g., organic acids, certain polar
pigments and sugars).
First and foremost, authors considered the importance of sample
size and correct comminution. The selection of a representative test
portion of the sample is essential to ensure that significant results
are obtained. Similarly, samples should be properly comminuted to
maximize surface area, ensuring better extraction efficiencies during
shaking. Thus, they selected 10 g of sample based on previous ex-
perience and evidence in the literature. Different representative
sample sizes can be used (normally 10–15 g) if the method is ap-
propriately scaled and the sample is properly comminuted.
Once sample size and comminution were fixed, conditions for
extraction and clean-up were assessed. It is worth mentioning that
decisions were made as compromises to maximize simplicity, speed,
selectivity and recoveries. On that basis, a wide variety of factors
were evaluated, including sample constitution, type of extraction
solvent, sample/solvent ratio, type and time of extraction proce-
dure (blending or shaking), extraction temperature, addition of non-
polar co-solvents and/or salts and materials used for clean-up.
Acetone, ACN and EtOAc were selected for extraction, since they
were the most commonly used for the multi-residue analysis of pes-
ticides. ACN was able to extract pesticides with a wide range of
polarities with higher capacity and selectivity than the other two
solvents evaluated. Moreover, suitable miscibility with water (as in-
dicated by the authors, the water content of most fruits is in the
range 80–95%) allowed good penetration into the aqueous frac-
tion of samples, also permitting a relatively easy separation of the
different phases by adding salts (avoiding the need for non-polar
solvents). Also, the extraction of lipophilic material, waxes, fats and
pigments was greatly reduced in comparison with the other sol-
vents tested. In addition, the authors pointed out that ACN offered
the possibility of a subsequent liquid-liquid clean-up step, if needed
for other applications, since it forms distinct partitioning phases with
non-polar solvents. However, the d-SPE procedure was preferred
in most subsequent applications. For these reasons and despite the
inherent disadvantages of ACN (i.e., larger solvent expansion volume
during vaporization in GC, lower volatility, greater cost and higher
toxicity), this solvent was selected. Regarding solvent/sample ratio,
a proportion 1:1 was shown to be sufficient and adequate to achieve
a quantitative extraction.
Concerning the use of salts to induce phase separation, MgSO4,
MgCl2, NaNO3, NaCl, Na2SO4, LiCl and fructose were tested {the use
of drying salts (e.g., Na2SO4 or MgSO4) to improve recoveries of polar
compounds had already been reported on a good number of occa-
sions [10–12]}. Among the studied salts, MgSO4 provided the most
complete liquid-liquid phase separation and was better able to bind
large amounts of water. As a consequence of the exothermic reac-
tion of hydration, the extraction of pesticides (especially non-
polar) was favored by temperature, which could reach 40–45°C.
Moreover, the advisability of using MgSO4 and NaCl separately or
in combination was studied. Although recoveries were not satis-
factory when NaCl was used alone (or in greater amounts in
combination with MgSO4), a more complete phase separation was
produced by adding NaCl. Also, and interestingly, the introduction
of NaCl allowed the amount of matrix components co-extracted to
be reduced, as can be seen in Fig. 1, in which reduction of fructose-
degradant product HMF (which occurs in the GC injector) was
observed with increasing NaCl. Moreover, the amount of NaCl used
also had a great influence on the peak shapes and areas of several
pesticides. For these reasons, NaCl was included.
Concerning sample pH, it should be taken into account that the
pH values of vegetables and fruits are in the range 2–7. Based on
several pesticides used in agriculture being less stable at basic pH
and that others can be lost at low pH, authors studied the effect of
pH by analyzing the recovery values obtained after adjusting the
acidity of an apple juice to 2.5, 3, 4, 5, 6 and 7. Since water is also
present in the ACN phase, authors hypothesized that basic recov-
eries of pesticides should not be greatly reduced at low pH. They
found that the effect was negligible and acidic pH values were used.
Samples with intrinsic low pH could be used with no modifica-
tion of pH, but samples with higher values were adjusted to pH lower
than 4 to minimize degradation. Moreover, it was found that pH
could affect the amount of co-extracted compounds. According to
 M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185
Fig. 1. GC-MS chromatograms (full-scan mode) of apple extracts obtained by using
different amounts of NaCl with 5 g of MgSO4 in the phase separation between water
and ACN for the extraction of three pesticides (methamidophos, acephate, and
omethoate). {Reprinted from [1] with permission of AOAC International}.
the results obtained, the quantity of certain acids (including fatty
acids) increased as the pH decreased. Thus, the amount of salt added
should be cautiously optimized to reduce this co-extraction. Finally,
not only the pH value of the sample but also the pH of the ACN phase
could affect the stability of pesticides (PSA increases the pH of the
extracts). The authors observed that alkaline-sensitive pesticides were
stable in ACN containing 0.05–0.1% HOAc but they indicated that
future experiments should be made to evaluate the usage of a small
amount of HOAc in ACN for the entire extraction.
The clean-up and drying of the ACN phase were performed si-
multaneously at the d-SPE step. The selection of the ideal sorbent
was based on the determination of co-extracted materials removed
by the sorbents evaluated (N-propylethylendiamine or PSA, a
methacrylate-divinylbenzene copolymeric sorbent, GCB, alumina-
neutral, a strong-anion exchanger, cyanopropyl, aminopropyl and
C18) in the ACN extract. PSA did not retain the pesticides but it ef-
fectively removed polar interferences (including fatty acids, other
organic acids, sugars and pigments, such as anthocyanidines),
whereas GCB efficiently eliminated pigments (e.g., chlorophyll and
carotenoids), but showed strong affinity towards planar mol-
ecules (including some pesticides), which clearly reduced the
recovery values.
Regarding the addition of MgSO4 as drying salt to the ACN phase,
it proved to be advantageous because it provided less polar ACN ex-
tracts, and residual water could have an effect on both the d-SPE
procedure and GC separation. Besides, certain polar co-extractives
were also precipitated in some samples, since peaks belonging to
polar matrix components were partly obscured in the
chromatograms.
As a result, the optimized original method consisted of the ex-
traction of 10 g of sample with 10 mL of ACN followed by the
addition of 4 g of MgSO4 anhydrous and 1 g of NaCl, which were
immediately mixed by vortex to prevent formation of MgSO4 con-
glomerates. The ratio 1:4 of salts was shown to be the most effective
in selectivity and in capacity to separate aqueous and organic phases,
maintaining high recoveries and low co-extraction of interfer-
ences. The sample was centrifuged at 5000 rpm for 5 min. Then,
25 mg of PSA and 150 mg of MgSO4 were added to 1 mL of super-
natant and the mixture was shaken by hand (or vortex) for 30 s. After
a second centrifugation at 6000 rpm for 1 min, the extract was ready
for analysis in the GC-MS system. At that moment, authors already
advanced one of the clear advantages of the method: a single analyst
171
could be capable of preparing a batch of 6–12 samples in 30–
45 min with a cost of approximately $1 (US) per sample.
This detailed manuscript, with nearly 20 printed pages, also
studied the application of analyte protectants and discussed in depth
the approach studied, already advancing its applications to other
pesticides, matrices and LC techniques.
3. Most relevant modifications
Although the original method was shown to be efficient for hun-
dreds of analytes in a wide variety of commodities, as it was very
rugged, particularly for pesticides in food matrices [1,13], subse-
quent adjustments were developed in order to make the method
performance even better for some difficult analytes and commodi-
ties. The majority of these modifications were based on the
QuEChERS fundamentals using the two different steps, but modi-
fying the extraction solvent and the salt and sorbent formulations
of the salting-out and the d-SPE steps. Many of these modifica-
tions emerged from the need to obtain high recovery values in
matrices of different complexity, to avoid pesticide degradation and
to diminish the matrix effects. As evidence, several companies offer
to accommodate the many preferences of the customers. In any case,
there are three main modifications that are part of what can be seen
today as the universal QuEChERS method.
The first and perhaps most relevant modifications were devel-
oped to expand the method to certain pesticides that undergo
ionization and/or degradation during extraction, depending on the
pH of the matrix. So, the original unbuffered version [1] evolved into
two official methods using citrate buffering at a relatively low buff-
ering capacity {CEN Standard Method EN 15662, developed by
Anastassiades and co-workers [14]} or acetate buffering at higher
concentration to give a greater buffering strength {AOAC Official
Method 2007.01, developed by Lehotay [15]}. Both versions lead to
a pH around 5, which corresponds to a compromise to extract sat-
isfactorily those pesticides that are sensitive under acidic or basic
conditions (e.g., folpet, dichlofluanid, pymetrozine, chlorothalonil),
regardless of the matrix. These versions have been widely evalu-
ated and adopted as routine methods in many laboratories around
the world. Nevertheless, buffering is not recommended for some ma-
trices (e.g., those with a high lipid content), since the amount of
co-extractives increased due to the reduced capacity of PSA to retain
them at such pH [16].
Secondly, concerning d-SPE clean-up, other modifications in-
cluded the use of different amounts of PSA [16] or C18 together with
PSA to obtain cleaner extracts. The use of C18 is particularly effec-
tive for those samples with relatively high lipid content {e.g., cereals
[17,18]} and it does not negatively affect pesticide recoveries, con-
trary to what happens with the use of greater amounts of PSA {i.e.,
~20% lower recoveries for some polar pesticides [16]}. Hence, some
modified methods have used only C18 to remove fats since PSA was
not necessary or decreased the recoveries [19].
Thirdly, GCB has also been combined with PSA to remove chlo-
rophyll co-extractives from green matrices (e.g., lettuce, spinach or
leaves), resulting in less colored extracts [18,20]. However, GCB is
well known to sacrifice ~25% of the recoveries of certain pesti-
cides with planar functionality (e.g., hexachlorobenzene,
thiabendazole, and terbufos), which tend to be retained on its surface
[21].
There was a certain need to harmonize the amount of these pa-
rameters for a universal QuEChERS method. In this sense, comparison
of the main QuEChERS versions indicated that acetate buffering for
the extraction of 15 g of homogenized sample (ground by adding
dry ice to produce a flowing powder) using 15 mL of 1% HOAc in
ACN and 6 g of MgSO4 anhydrous + 1.5 g of NaOAc, and using 50 mg
of PSA + 50 mg of C18 + 7.5 mg of GCB (plus 150 mg of MgSO4 an-
hydrous as drying agent to reduce the amount of water from ≈7%
172 M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185

to ≈2% in the final extracts which reduces the amount of water being
introduced into the GC system) per mL of extract for clean-up pro-
vides overall the most beneficial sample pretreatment for the analysis
of pesticides in fruits and vegetables [22]. For cereals and grains,
2.5 g or 5 g of sample (plus 10 mL of water to weaken the possible
interactions between analytes and matrix, improving the extrac-
tion) is extracted and 150 mg of PSA is used, rather than 50 mg [16].
From here, many modifications have been developed beyond simple
changes in the amounts of sample, solvent, salt or sorbents. The fol-
lowing are some of the most significant modifications.
Regarding the extraction step, González Curbelo et al. [23] de-
veloped and evaluated three new different versions of the QuEChERS
method based on the use of ammonium chloride and ammonium
formate and acetate buffers to induce phase separation and extrac-
tion of representative pesticides. Such salts were used as alternatives
to MgSO4 and sodium salts, since the latter tend to deposit as solids
on surfaces in the MS source and potentially within the analyzer
or in the inlet liner in GC to cause loss of instrument performance
and to require increased maintenance. Ammonium salts did not pose
a problem in the ion source because they decomposed at typical
ion-source temperatures. Also, ammonium can enhance the for-
mation of analyte ions instead of undesirable sodium adducts.
Although the performance of the three methods compared favor-
ably with QuEChERS in the AOAC Official Method 2007.01, the use
of the formate buffer [using 7.5 g of ammonium formate and 15 mL
of 5% (v/v) formic acid in ACN for the extraction of 15 g of fruit/
vegetable samples] ensured a suitable pH for high recoveries of most
pesticides independently of the matrix. Moreover, method valida-
tion was carried out with and without the use of the d-SPE clean-
Fig. 2. Freezing block consisting of a laboratory rack with 15-mL centrifuge tubes
up but no significant differences were observed for the majority of
immersed in a polystyrene box filled with freezing gel. {Reprinted from [31] with
pesticides. This new version of the QuEChERS method really aims permission from Elsevier}.
to substitute for previous ones, as a result of the inherent advan-
tages of using ammonium salts instead of sodium salts, which are
not desirable in MS analysis. Also, this ammonium-formate buff-
ering is most promising for wide applicability of LPGC-MS/MS and mersed in the ACN phase and the extract was aspirated. Then,
LC-MS/MS (also FI-MS/MS) analysis for pesticides in foods and pos- syringes were inserted in the block previously introduced in the
sibly other analyte/matrix combinations. Recently, Han et al. [24] freezer for 2 h. After freezing, the piston was pressed and the filter
also applied with success ammonium-formate buffering to the ex- retained the precipitated material.
traction of 42 diverse pesticides and 17 environmental contaminants, For the same purpose, and more recently, new sorbent materi-
including PAHs, PCBs and flame retardants from shrimps. als (e.g., based on zirconium oxide) were also used [32,33]. There
The effect of temperature on the extraction of thermally-labile are currently two variants of zirconium oxide available: so called
analytes has also been evaluated. Some pesticides can be de- Z-Sep Plus and Z-Sep. The first is silica coated with ZrO2 while the
graded with the addition of MgSO4 anhydrous due to its exothermic second also has C18 in proportion 2:5. Another new type of sorbent,
hydration reaction [25]. Freezing the sample before extraction [26] called ChloroFiltr, is being used for the selective reduction of chlo-
or adding cold water (<4°C) [27] reduces this negative effect. In par- rophyll from green-plant extracts [34]. In this case, the use of 50 mg
ticular, when using frozen samples (as generally advised), the per mL of extract provides clean extracts without a significant de-
temperatures reached after extraction are very moderate. By con- crease in recoveries. Other modifications include the addition of
trast although the change in the purity of MgSO 4 achieves a MWCNTs [35], magnetic nanoparticles [36], alumina [37], and Florisil
remarkable temperature difference, it does not lead to significant [38].
differences in recoveries [28]. Finally, other versions of the clean-up step, which consists of re-
The d-SPE step has also been combined with the so called freeze- placing the d-SPE procedure by a SPE column, have been developed.
out step, which consists of the precipitation of lipids at low For this, columns of aminopropyl (-NH2) and PSA [39] and columns
temperature [27,29]. However, although it does not require the use of GCB and PSA [40] have been used. In both cases, it was found
of additional sorbents, it clearly increases the sample-pretreatment that, although the SPE procedure is more effective and has greater
time because freezing time is ~1–2 h. Moreover, it was well dem- cleaning power for the final extract, the simplicity and the speed
onstrated for pesticide analysis that it is unnecessary when it is of the d-SPE procedure are more decisive in routine analysis, so d-SPE
carried out after the d-SPE step using PSA and C18. In this case, the remains the most widely used mode in most applications. Further-
amount of co-extractives obtained is equivalent to that obtained more, the MgSO4, which is extremely important to reduce water
when including freezing out [30]. Even so, Norli et al. [31] found content in the final extracts, tends to clog the frits [25], so the op-
that the extraction of 22 OCPs and seven PCBs from fish (tilapia and erational level of d-SPE remains more effective than SPE.
salmon) benefitted from this procedure. For this purpose, and in
order to improve sample throughput, they developed equipment 4. Application fields
comprising disposable syringes and a freezing block consisting of
a laboratory rack with 15-mL centrifuge tubes immersed in a poly- As mentioned above, the application of QuEChERS has widely
styrene box filled with freezing gel (Fig. 2). After solvent extraction, expanded from the pesticide-analysis field. Tables 1–5 summarize
disposable syringes with polyethylene frits in the bottom were im- some examples of its applications, considering only those works in
Table 1
Some examples of the application of the QuEChERS method to the analysis of pesticides

Analytes Sample (amount) Extraction Sorbents in the d-SPE step Analytical technique Recovery (%) LODs Comments Ref.

Solvents Salts

M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185

229 pesticides Lettuce and 15 mL ACN 6 g MgSO4, 1.5 g NaCl 1.8 g MgSO4, 300 mg PSA HPLC-MS/MS and 70–120a <10 μg/kg (HPLC- TPP as IS [13]
orange (15 g) GC-MS MS/MS) and 30–
100 μg/kg (GC-MS)
43 pesticides Apple, lemon, 10 mL ACN 5% 7.5 g ammonium formate 150 mg MgSO4, 50 mg PSA LPGC-MS/MS 90–110 <5 μg/kg Atrazine-d5 and fenthion-d6 as [23]
lettuce (15 g) and HOAc (v/v) (150 mg for wheat), 50 mg C18, ISs; commercial QuEChERS kits
wheat grain (5 g) 7.5 mg GCB
34 pesticides Flaxseeds, peanuts 10 mL ACN 4 g MgSO4, 1 g NaCl 150 mg MgSO4, 150 mg PSA, GC-MS 70–120a 0.005–0.3 μg/kg 10 mL of water were added [30]
and doughs (2.5– 50 mg C18 initially; 6 isotopically labeled
5 g) ISs; commercial QuEChERS kits
15 pesticides Milled toasted 10 mL ACN 0.5% 4 g MgSO4, 1 g NaCl 750 mg MgSO4, 800 mg PSA, GC-NPD 71–110 0.07–34.8 μg/Kg 10 mL of water were added [41]
wheat and maize, formic acid (v/v) 31 mg C18, 274 mg GCB initially; TPP as IS
wheat flour and
baby cereals (5 g)
77 pesticides Red and white 5 mL ACN 4 g MgSO4, 3 g NaCl 150 mg MgSO4, 50 mg PSA GC-MS 70–110 0.9–15 μg/L TPP as IS [42]
wine (10 mL)
73 pesticides Edible oil, meat, 14 mL ACN 1% 6 g MgSO4, 1.5 NaOAc, 900 mg MgSO4, 150 mg PSA, HPLC-MS/MS 70–120 3 μg/kg C18 only for fatty samples and [43]
egg, cheese, HOAc (v/v) 4 g NaCl 150 mg C18, 45 mg GCB GCB only for non-fatty but
chocolate, coffee, colored samples; TPP,
rice, tree nuts, omethoate-d6, propamocarb-
citric fruits, d7 and BDMC as ISs
vegetables,
beverages, seafood
(10 g)
143 pesticides Fish (10 g) 10 mL ACN 4 g MgSO4, 1 g NaCl 150 mg MgSO4, 50 mg Z-Sep LPGC-MS/MS 70–120a 0.5–5 μg/kg Atrazine-d5 and fenthion-d6 as [44]
ISs
36 pesticides Soil (5 g) 10 mL ACN 6 g MgSO4, 1.5 g NaCl, 1.5 tri- 150 mg MgSO4, 150 mg PSA, GC-MS 70–120a <7.6 μg/kg 3 mL of water were added [45]
Na, 0.75 g di-Na 50 mg C18 initially; 4,4’-
dichlorobenzophenone as IS;
commercial QuEChERS kits
6 pesticides Human blood and 1 mL ACN 0.4 g MgSO4, 0.1 g NaAc 150 mg MgSO4, 25 mg PSA, HPLC-MS/MS 87–112 0.46–1.15 μg/L d10-disulfoton as IS; [46]
urine (0.5 mL) 25 mg C18 commercial QuEChERS kits
a
Recovery values were found at 70–120% for most of analytes determined.

173
174 M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185
which a full version of the QuEChERS method has been applied (some
recent and relevant works concerning pesticides have also been in-
cluded in Table 1).
4.1. Pesticide analysis
The determination of pesticides, especially in food matrices, is
a topic of extraordinary relevance. They are the compounds for which
a higher number of regulations/controls have been developed in
order to cover the types and the quantities of pesticides to which
the population is exposed. That is why many countries have in-
cluded these analytes as pollutants, hazardous to the human health.
In this sense, diverse organizations have established MRLs in fruits,
vegetables, cereals, foods of animal origin and water. The EU, for
example, legislates values in the range 0.01–10 mg/kg for the adult
population and even lower concentrations for baby foods in the range
0.003–0.01 mg/kg, which highlights the importance of the appli-
cation of an efficient sample treatment to carry out their
determination in these complex samples. For many years, the
methods most commonly used, which were in general quite tedious,
required the utilization of large amounts of toxic organic solvents
[47]. The introduction of the QuEChERS method clearly provided
a fast effective alternative that soon replaced most of the previous
approaches, especially because of the significant elimination of
matrix components and obtaining high recoveries of pesticides with
a wide range of polarities.
The excellent results achieved for fruits and vegetables soon led
to the application of QuEChERS to other food matrices, especially
multi-class pesticide analysis, which has increased in recent years
[8,18,48]. As indicated above, AOAC Official Method 2007.01 [15] and
CEN Standard Method EN 15662 [14] are without any doubt pre-
ferred in the field.
Regarding other food matrices (see Table 1), cereals [23,30,41],
alcoholic drinks [42], dairy products, meat [43], and fish [44] have
been studied, among others. As an example, Sapozhnikova [44] de-
veloped a methodology for the determination of 143 different
pesticides in fish using the original QuEChERS method with 50 mg
of Z-Sep (for 10 g of fish) instead of PSA as the clean-up sorbent.
Atrazine-d5 and fenthion-d6 were used as ISs. Good results were ob-
tained with LODs of 0.5–5 μg/kg and recoveries of 70–120%
(RSDs < 20%) for almost all analytes and significant removal of
coextractive material by reducing the matrix effect.
Although less extensively applied, QuEChERS has determined pes-
ticides in biological fluids or non-edible plants and in environmental
matrices (e.g., water, soil or sediments) [8,9]. Regarding sedi-
ments, this use constitutes a very recent approach, since this complex
matrix also involves biological, physical and chemical processes.
The application of QuEChERS to environmental matrices was done
with the original methodology, but, in the majority of cases, diverse
modifications were introduced. In this way, Fernandes et al. [45]
studied the application of different modified versions of QuEChERS
based on the citrate-buffer method for the determination of 36 pes-
ticides in organic farming and integrated pest management soils.
The modifications focused on the amount of sample used, the sor-
bents required in the clean-up step and the volume of water added
at the beginning of the extraction in order to improve recoveries.
Besides, a more dramatic change was applied by inclusion of ul-
trasound assistance in the extraction step. This modification, which
was applied on many occasions for the determination of pesti-
cides in soils using QuEChERS methodology, provided a good
homogenizing process and, consequently, better extraction effi-
ciencies. In this work, the best results were obtained with 0.5 g of
soil and 3 mL of water extracted with 10 mL of ACN, and 6 g of
MgSO4, 1.5 g of NaCl, 1.5 of tri-Na and 0.75 g of di-Na. After adding
salt, ultrasound was applied for 5 min and then centrifugation. Af-
terwards, sample clean-up was carried out with 150 mg of MgSO4,
150 mg of PSA and 50 mg of C18 as salts, vortexing for 1 min and
centrifugation. Finally, 500 μL of the upper layer was injected into
the GC-MS system, obtaining LODs lower than 7.6 μg/kg and re-
coveries of 70–120% for almost all analytes. In this case d-SPE clean-
up efficiency was compared with the same process carried out with
a disposable pipette extraction, and similar results were obtained
with both procedures.
Concerning the analysis of bioanalytical samples, the number of
applications remains low and only a low number of pesticides
studied. An example is the work of Usui et al. [46], who carried out
the extraction of disulfoton and five of its metabolites in 0.5 mL of
human whole blood and urine using 1 mL of ACN and 0.5 g of a
SampliQ QuEChERS kit (AOAC), which contains 6 g of MgSO4 and
1.5 g of NaOAc. After hand shaking for 30 s and centrifugation, the
d-SPE procedure with the same commercial kits, containing 150 mg
of MgSO4, 25 mg of PSA and 25 mg of end-capped C18, was applied.
Good results were obtained with LODs of 0.46–1.15 μg/L and re-
coveries of 87–112%. The validated methodology was applied to a
real forensic case, revealing the presence of disulfoton and its me-
tabolites in the test sample.
Regarding ISs, although isotopic labelling of target pesticides is
commonly applied [23,30,43,44,46], others {e.g., TPP [13,41,43],
which is the most common, BDMC, 4,4’-dichlorobenzophenone,
diazinon or ethoprophos} have been used occasionally for specific
groups of pesticides. However, other compounds (e.g., PCBs), which
were initially proposed as potential ISs, are not usually applied. In
general, introduction of an IS is highly recommended at the begin-
ning of the process, except for samples with higher fat content, since
the solubility of fat in the ACN layer is very limited, generating an
additional fat layer where analytes could partition and get lost [49].
Regarding the analytical methods applied for determination of
pesticides, as is usual since the introduction of QuEChERS, LC-MS/
MS and GC-MS/MS {also LPGC [23,44] or GCxGC [50]} are the most
common systems used, Q-MS and QqQ-MS/MS being the analyz-
ers most commonly used [9]. Concerning CE, though not established
as a routine analysis in the field, some works have also demon-
strated the application of CE for pesticide analysis in combination
with the QuEChERS method [51].
Despite QuEChERS providing excellent results for a large number
of pesticides, the method is still sometimes criticized for not being
able to analyze all pesticides completely. Some examples are those
of the “quat” family, glyphosate and its metabolite AMPA or
chlorothalonil, which have to be analyzed by single-class or single-
analyte methods. Other examples are folpet, chlorothalonil, captan
or captafol, which are only amenable to GC with certain problems
(though the use of analyte protectants improves their analysis) and
are easily degraded in non-acidic samples (acid addition reduces
the losses but not as much as required). By contrast, others (e.g.,
carbosulfan or benfuracarb) are not protected sufficiently at acid
pH and it is necessary to obtain a non-acidified extract after the
clean-up step in order to maintain their stability [52].
4.2. Pharmaceutical analysis
Use of pharmaceutical compounds is being abused, especially
in veterinary treatment to control diseases and to promote the
growth of livestock. These practices, if not properly controlled, may
constitute significant risks to human health and the environment.
For this reason, many pharmacologically-active substances have been
considered pollutants, and their use has begun to be controlled by
different organizations establishing MRLs for many of them. As an
example, the EU has established MRLs of 0.05–20,000 μg/kg for
pharmacologically-active substances in foodstuffs of animal origin;
these values may be different for a given analyte, depending on the
type of livestock or tissue to be analyzed [53]. As a consequence,
pharmaceutical-compound analysis is also an active analytical area,
 M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185
in which fast feasible analytical methods are required, especially
in food control and safety. In this sense, the application of the
QuEChERS method was demonstrated to be successful (see Table 2
for some examples), for the separate determination of certain fami-
lies of drugs {e.g., sulfonamides [54], quinolones [55], anthelmintics
[19,56–59], or benzimidazoles [60]} and to carry out multi-residue
analysis [61–65].
As usual, the separation technique most widely applied for phar-
maceutical compounds analysis in combination with QuEChERS is
LC, both HPLC [54,61,66] and UHPLC [56–60,63,64]. Even capillary
LC [55] or GC (less common) [65] have been selected. In most cases
and as desirable, these techniques have been coupled to MS detec-
tion systems [19,54,56–61,63–66]. In certain cases, the determination
of the target analytes was carried out without previous chromato-
graphic separation by DART [62] – for example, the work of Martínez-
Villalba et al. [62], who carried out the determination of a group
of 20 benzimidazoles in milk and four coccidiostats in feed – for
that, their modified QuEChERS procedure consisted of an extrac-
tion with ACN for feed samples and ACN 0.1% (v/v) NH3 for milk and
the addition of a mixture of MgSO4 and NaCl. The clean-up step con-
sisted of adding MgSO4, C18 and PSA. This new methodology obtained
good recovery percentages (65–95%) and demonstrated the possi-
bility of quantifying a variety of veterinary drugs at the levels
established by the EU legislation [53].
The samples analyzed more often have been those of animal
origin, probably because of the risk of accumulating these com-
pounds in their tissues and their subsequent risk for human beings.
They include different animal tissues [19,54,56,57,59,61,63,64], milk
[19,55,58,60,62] or blood [65]. However, other matrices {e.g., animal
feed [62,66]} have also been analyzed to prevent contamination of
animals via food ingestion.
As can be seen in Table 2, different versions of QuEChERS have
been used to achieve effective extraction of the target analytes. Re-
garding pH control/adjustment, which is probably the main difference
between methods, some papers have included acid addition {e.g.,
formic [55] or HOAc [54,61,63,64,66]}, bases {e.g., NH3 [60,62]} or
even buffering at a given pH with buffer solutions [55] or addition
of salts {i.e., sodium acetate [66] or citrates [54,55], according to
the AOAC Official Method 2007.01 and EN 15662 norm, respective-
ly}, depending on the nature of the target analyte.
Regarding the clean-up step, few different options can be found
in the literature. In this sense, C18 has been used for removal of fats
and hydrophobic compounds in muscle tissue [56,57,61], milk
[19,55,58,60,62], feed [62] and liver [19,59], in most cases, in com-
bination with PSA [60,62] (the only sorbent used apart from C18).
On other occasions, PSA has been used alone [54,63–66] {e.g., in ex-
traction of certain animal tissues [54,63], shrimp [64], animal feed
[66] or blood [65]}. In most cases, both sorbents are used in com-
bination with MgSO4, although it is possible to find a few works
where MgSO4 was not added [61,63,66].
Generally, QuEChERS methods developed for the analysis of phar-
maceutical compounds have provided recovery percentages of 70–
120% except for certain analytes. However, it should be noted that,
in many cases, ISs are also used to correct the losses that occur during
the extraction procedure and that calibration is also developed in
the matrix. In most works, and, as usual, deuterated ISs have been
used [19,54,56–60,62]. Moreover, it should be noted that some
studies have also applied commercial QuEChERS kits [19,54–59],
which, in many cases, were designed for the extraction of pesti-
cides, showing the versatility of the procedure. Finally, most of these
methodologies have provided LODs in the low-μg/kg range.
4.3. Mycotoxin analysis
Mycotoxins are secondary metabolites produced by many species
of fungi and constitute an important group of contaminants, espe-
175
cially in food. These compounds can cause significant problems with
adverse estrogenic, nephrotoxic, hepato-toxic, neurotoxic or im-
munosuppressive effects and even carcinogenesis [67], so effective
sample-pretreatment steps (e.g., QuEChERS) are necessary in order
to carry out exhaustive controls and to avoid their hazardous con-
sequences for human health.
The determination of this group of analytes of great concern has
been developed exclusively in food analysis [67–75], since food is
the main route for human exposure. Cereals are one of the matri-
ces in which QuEChERS has been most commonly applied and
extraction of mycotoxins is no exception – important articles have
focused on the extraction of wheat [68,69,72,76], maize [69,72,75,76],
soy [69], oat [69], rice [69,70], corn [75,77], and millet [72]. Table 3
shows a representative group of QuEChERS procedures, with sample
amounts of 2–15 g being usual, and, since cereals have very low per-
centages of water, addition of water has also been needed at the
beginning of the process [68–70,72–77].
Although cereals constitute the most important matrix regard-
ing mycotoxin extraction using QuEChERS, the presence of these
analytes in other matrices {e.g., dairy products [67] or food supple-
ments} has also been studied [71]. In this sense, Jia et al. [67] carried
out an extensive study of the presence of 58 mycotoxins in dairy
products (e.g., milk, milk beverages and yogurt). For this purpose,
the initial extraction of 15 g of each sample with 10 mL of 84/16
(v/v) ACN/H2O containing 1% (v/v) of HOAc was developed. After
1 min of vortexing, 6 g of MgSO4 and 1.45 g of NaOAc were added
in order to induce phase separation. Then, the mixture was shaken
and centrifuged at 4°C. The use of the acetate buffer improved sta-
bility and recovery of certain pH-dependent compounds (e.g., patulin
or ZEN). For the clean-up step, 1.2 g of MgSO4, 108 mg of PSA and
405 mg of C18 were added to 8 mL of the upper layer, vortexed and
centrifuged again at 4°C. Then, 200 μL of the extract were filtered
after dilution with MeOH and 8 mM ammonium-formate buffer. This
solution was injected into an UHPLC-ESI Q-Orbitrap system. Re-
coveries of 87–114% (RSDs < 6.2%) and LODs of 0.001–0.92 μg/kg
demonstrated the enhancements of sensitivity and accuracy in com-
parison with traditional methods. Together with the simplicity of
the extraction, this approach showed a powerful, fast method for
the rapid screening of mycotoxins in foods.
Regarding the extraction step, mycotoxin analysis has also re-
quired the addition of HOAc [74] or formic acid [73], citrate salts
[70,73] or NaOAc [67,74], in order to avoid increasing pH after adding
PSA in the second step. In general, C18 and PSA are, again, the sor-
bents most commonly applied in d-SPE, but the combination of these
two with Al-N also showed good results [70]. C18 has been used alone
for the extraction of cereals [68,69,73,76], while PSA has been used
without C18 for the extraction of dairy products [67], bee-pollen-
based food supplements [71] or cereals [67,70,71,74]. As an example,
Fig. 3 shows the effect on the DART-MS signal of mycotoxin DON
of the amount of PSA added to the ACN cereal extract, as devel-
oped in the work carried out by Vaclavik et al. [72]. Judging from
Fig. 3, the positive effect on the signal is apparent from the addi-
tion of this sorbent (when direct MS analysis is developed, it is clear
that sample pretreatment should provide extracts as clean as
possible).
Concerning analyte quantification, apart from the use of
isotopically-labelled ISs [72,75,76], other compounds {e.g., pheno-
thiazine [77] or ZEN [76]} have also been selected with success.
As usual, some studies of mycotoxin analysis were also carried
out with the aim of comparing QuEChERS with other extraction
methods. Rodríguez-Carrasco et al. [68] carried out a comparison
between MSPD and QuEChERS for the extraction of eight trichotecene
mycotoxins from wheat-based breadstick snacks followed by GC-
MS/MS analysis. They found that QuEChERS extraction is faster
and less laborious, and shows better extraction efficiencies (recov-
eries higher than 80% in most cases) than MSPD (recoveries of
 176
Table 2
Some examples of the application of the QuEChERS method to the analysis of pharmaceutical compounds

Analytes Sample (amount) Extraction Sorbents in the Analytical Recovery (%) LODs Comments Ref.
d-SPE step technique
Solvents Salts

38 anthelmintic Bovine milk and 10 mL ACN 4 g MgSO4, 1 g NaCl 150 mg MgSO4, LC-MS/MS 70–120a 11–112 μg/kgb TPP and 2,4-D as ISs; [19]
drug residues liver (10 g) 50 mg C18 for milk commercial QuEChERS kit
11–1721 μg/kgb
for liver
22 sulfonamides Edible beef, sheep, 10 mL ACN 1% 0.5 g di-Na, 1 g tri-Na, 4 g 150 mg PSA, HPLC-HRMS 88–112 for 3–26 μg/kg 5 mL of water were added [54]
and their chicken and pig HOAc (v/v) MgSO4, 1 g NaCl 900 mg MgSO4 beef muscle initially;
metabolites tissues (5 g) SMX-D4 as internal standard;
commercial QuEChERS kit
7 quinolones Milk (2 g) 10 mL ACN 5% 4 g MgSO4, 1 g NaCl, 1 g 150 mg C18, 900 mg MgSO4 Capillary LC-LIF 83–104 0.4–6.0 μg/kg 8 mL of 30 mM NaH2PO4 buffer [55]

M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185

formic acid (v/v) tri-Na, 0.5 g di-Na pH 7.0 were added initially;
commercial QuEChERS kit
37 anthelmintic Bovine muscle 12 mL ACN 4 g MgSO4, 1 g NaCl 1.5 g MgSO4, 0.5 g C18 UHPLC-MS/MS - 0.15–10.2 μg/kgb 13 deuterated and 4 non- [56]
drugs and (10 g) deuterated ISs;
metabolites commercial QuEChERS kit
37 anthelmintic Bovine muscle 12 mL ACN 4 g MgSO4, 1 g NaCl 1.5 g MgSO4, 0.5 g C18 UHPLC-MS/MS 70–120a 0.15–10.21 μg/kgb 13 deuterated and 4 non- [57]
drugs (10 g) deuterated ISs;
commercial QuEChERS kit
38 anthelmintic Milk (10 g) 12 mL ACN 4 g MgSO4, 1 g NaCl 1.5 g MgSO4, 0.5 g C18 UHPLC-MS/MS 70–120a 0.18–123 μg/kgb 14 deuterated and 5 non- [58]
drugs deuterated ISs;
commercial QuEChERS kit
38 anthelmintic Liver (10 g) 12 mL ACN 4 g MgSO4, 1 g NaCl For low concentration levels UHPLC-MS/MS 70–120a Low concentration 14 deuterated and 5 non- [59]
drugs 1.5 g MgSO4, 0.5 g C18 levels deuterated ISs;
For MRL concentration level 0.16–1.66 μg/kgb commercial QuEChERS kit
150 mg MgSO4, 50 mg C18 MRL level
13–1593 μg/kgb
19 benzimidazoles Milk (10 g) 10 mL ACN 0.1% 4 g MgSO4, 1 g NaCl 150 mg MgSO4, 50 mg C18, UHPLC-MS/MS 70–120a - MBZ-d3 as IS [60]
NH3 (v/v) 50 mg PSA
55 multi-class Porcine, bovine and 16 mL ACN 5% 2 g NaCl, 4 g Na2SO4 400 mg C18 HPLC-MS/MS 70–120a - - [61]
veterinary drug ovine muscle HOAc (v/v)
residues tissues (4 g)
24 antiparasitic Feed and bovine 10 mL ACN (feed) 4 g MgSO4, 1 g NaCl 150 mg MgSO4, 50 mg C18, DART-MS 65–95 1–500 μg/kgc 10 mL of water were added [62]
veterinary drugs milk (10 g) or 10 mL ACN 0.1% 50 mg PSA initially;
(4 coccidiostats NH3 (v/v) (milk) MBZ-d3 as IS
and 20
benzimidazoles)
20 veterinary drug Chicken muscle 10 mL ACN:water 0.5 g di-Na, 1 g tri-Na, 4 g 150 mg PSA UHPLC-MS/MS 70–120a 3.2–16 μg/kg 5 mL of water were added [63]
residues (5 g) (80:20, v/v) 1% MgSO4 initially
HOAc (v/v)
13 multiclass Shrimp (10 g) 10 mL ACN 1% 4 g MgSO4, 1.75 g NaCl 250 mg PSA, 750 mg MgSO4 UHPLC-MS 70–120a 0.06–7.10 μg/kg - [64]
antibiotics and HOAc (v/v)
veterinary drugs
40 pharmaceuticals Blood (1 mL) 2 mL ACN 500 mg MgSO4, 250 mg NaCl 25 mg PSA, 25 mg MgSO4 GC-MS For 8 model For 8 model TPM as IS; the method was [65]
substances substances validated for only 8
90–104 5.6–17.2 μg/L compounds
13 sulfonamides Poultry and swine 10 mL ACN:MeOH 4 g MgSO4, 0.5 g NaOAc 200 mg PSA HPLC-MS/MS 86–107 0.5–4.2 μg/kg 10 mL of water were added [66]
feed (5 g) (75:25 v/v) 0.1% initially;
HOAc (v/v) sulfapyridine as IS
a
Recoveries values were found at 70–120% for most of analytes determined.
b Decision limit (CCα); c Lowest calibration level (LCL).
Table 3
Some examples of the application of the QuEChERS method to the analysis of mycotoxins

Analytes Sample (amount) Extraction Sorbents in the d-SPE step Analytical Recovery LODs Comments Ref.
technique (%)
Solvents Salts

58 mycotoxins Milk, milk 10 mL ACN:H2O 6 g MgSO4, 1.45 g NaOAc 1.2 g MgSO4, 405 mg C18, UHPLC-MS/MS 87–114 0.001–0.92 μg/kg - [67]
beverages and (84:16, v/v) 1% 108 mg PSA

M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185

yogurt (15 g) HOAc (v/v)
8 mycotoxins Wheat-base snack 8 mL ACN 4 g MgSO4, 1 g NaCl 900 mg MgSO4, 300 mg C18 GC-MS/MS 73–116 0.6–20 μg/kg 25 mL of water were added initially; [68]
(5 g) comparison with MSPE approach
3 mycotoxins Wheat, rice, maize, 7.5 mL ACN 4 g MgSO4, 1 g NaCl 900 mg MgSO4, 300 mg C18 GC-MS/MS 76–114 <10 μg/kg 25 mL of water were added initially [69]
spelt, oat, soy and
tapioca (5 g)
14 mycotoxins Rice (10 g) 10 mL ACN 10% 4 g MgSO4, 1 g NaCl, 1.2 g MgSO4, 250 mg C18, UHPLC-MS/MS 60–104 0.07–69.8 μg/kg Citrate QuEChERS; 10 mL of water [70]
formic acid (v/v) 1 g tri-Na, 0.5 g di-Na 400 mg PSA, 250 mg Al-N were added initially; comparison with
IACs approach
8 mycotoxins Bee pollen based 7.5 mL ACN 4 g MgSO4, 1 g NaCl 900 mg MgSO4, 300 mg C18, GC-MS/MS 73–95 1–4 μg/kg 10 mL of water were added initially [71]
food supplement 300 mg PSA
(5 g)
11 mycotoxins Wheat, maize and 10 mL ACN 4 g MgSO4, 1 g NaCl 600 mg MgSO4, 200 mg PSA DART-MS 84–118 50–150 μg/kga 10 mL of water were added initially; [72]
millet (2 g) 13C-deoxynivalenol, 13C-nivalenol and
13
C-zearalenon as ISs
26 mycotoxins Sesame butter 20 mL ACN:H2O 4 g MgSO4, 1 g NaCl, 900 mg MgSO4, 150 mg C18 UHPLC-MS/MS 60–120 0.05–6.94 μg/L - [73]
(2.5 g) (80:20, v/v) 0.1% 1 g tri-Na, 0.5 g di-Na
formic acid (v/v)
and 5 mL
ACN:H2O (80:20,
v/v) 0.1% formic
acid (v/v)
1 mycotoxin Cereal and animal 15 mL 6 g MgSO4, 1.5 g NaOAc 750 mg MgSO4, 250 mg C18, FIA-FD and FIA-TSL 86–112 1.5 μg/L (FD), 15 mL of water were added initially [74]
feed (15 g) ACN 0.1% HOAc 250 mg PSA 0.7 μg/L (TSL)
(v/v)
1 mycotoxin Wheat and corn 20 mL ACN 8 g MgSO4, 2 g NaCl 750 mg MgSO4, 125 mg PSA HPLC-MS/MS 92–108 5.8 μg/kg 20 mL of water were added initially; [75]
(5 g) 15-d1-deoxynivalenol as IS
5 mycotoxins Breakfast cereals 10 mL ACN 4 g MgSO4, 1 g NaCl 900 mg MgSO4, 300 mg C18 GC-MS 52–103 2–15 μg/kg 25 mL of water for cereals and 15 mL [76]
and wheat, maize for flours were added initially; sample
and cassava flours washing 2 times (5 mL n-hexane);
(5 g) zearalanone and 13C15-DON as ISs
1 mycotoxin Corn food (5 g) 15 mL ACN 5 g MgSO4, 1 g NaCl 750 mg MgSO4, 125 mg PSA HPLC-MS/MS 80 2 μg/kg 5 mL of water were added initially; [77]
phenothiazine as IS
a Lowest calibration level (LCL).

177
178 M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185
Fig. 3. Impact of the d-SPE clean-up employing PSA and MgSO4 on mycotoxin DON (m/z 331.0943 ± 4 ppm) signal intensity in wheat extract (spiked 500 μg/kg) obtained
after applying the QuEChERS method. {Reprinted from [72] with permission from Elsevier}.
46–89%). Besides, greater reproducibility was also found with RSDs
lower than 15% versus values higher than 37% for MSPD.
Koesukwiwat et al. [70] developed a similar comparison using
UHPLC-MS/MS with another approach: the use of IAC columns in
SPE. In this case, similar extraction efficiency was obtained for the
extraction of 14 mycotoxins from rice samples. However, al-
though acceptable extraction efficiency was obtained with IAC
(recoveries of 74–118%), a lack of sensitivity was found for the ma-
jority of the target analytes, with LODs higher than those obtained
with the QuEChERS method. Other disadvantages of IAC included:
higher complexity of the methodology, higher consumption of sol-
vents, higher costs, and, because of the high specificity of IAC, it was
impossible to carry out analysis of multiclass analytes (in contrast
with QuEChERS).
Concerning the separation techniques used, HPLC-MS/MS [75,77],
UHPLC-MS/MS [67,70,73] and GC-MS/MS [68,69,71] were used. In
this last case, derivatization after the d-SPE procedure using BSA/
TMCS/TMSI was necessary for the correct detection of mycotoxins.
Besides that, other minor analytical methods such as FIA with FD
and TSL [74] or DART-Orbitrap-MS [72] were used with good results,
though for a relatively small number of analytes.
4.4. PAH analysis
PAHs are a large group of organic environmental contaminants
that have been included by the EU and US EPA as priority pollut-
ants to be monitored, due to their recognized mutagenic and
carcinogenic properties. Concerning their extraction using the
QuEChERS method, most of the published works in the literature
have analyzed the 16 PAHs included in the US EPA priority pollut-
ants list [78–84] while others have determined only some of them
[85–90], the analysis of PAH metabolites [e.g., monohydroxylated
(OH-PAHs)] being less frequent [91]. For OH-PAHs, Knobel et al. de-
termined a group of five of them in cattle milk because of their
relevance as exposure biomarkers, employing capillary-zone elec-
trophoresis with UV detection for their analysis [91]. This work is
one of the few in the literature combining QuEChERS with CE
analysis.
As well known, there are a wide variety of PAH sources, so there
is a wide distribution of these compounds in the environment. In
addition to their lipophilic nature, this wide distribution leads to
a higher risk of contamination in food of animal origin. Thus, the
type of analyzed matrices in the literature varies a lot and in-
cludes different types of meat [78,81,83,85], milk [91], fish [89,90]
and seafood [78,79,82,84,86,88]. However, the method has also been
applied to PAH analysis in some foods of vegetable origin that may
also be contaminated during their drying process with wood burning
[80,87]. Moreover, since the same contamination may occur when
food is cooked in a grill using wood, the method was also applied
with success by Kao et al. to the extraction of PAHs in kindling free-
charcoal grilled meat and seafood [78].
Table 4 shows that there are not many variations with respect
to the extraction step in the QuEChERS procedure. A few works have
applied the reagents employed in the original method [79,82,84,86],
using ACN as solvent and MgSO4 and NaCl as extraction salts and
PSA for the clean-up step, though others replaced NaCl by NaOAc
in the same ratio [78,81,83,88,90].
In order to obtain good recovery values, some authors have
suggested changing the organic solvent {e.g., Surma et al. [85],
who used EtOAc to extract 12 PAHs from ham}. Perhaps, the most
striking variant was developed by Forsberg et al. [89] for the
extraction of 33 PAHs from fish. In this work, they used a mixture
of organic solvents (acetone, EtOAc and isooctane) because of its
improved selectivity for non-polar PAH residues and lower cost
than ACN. Besides using this mixture of solvents, they also ob-
tained good results using the salts indicated in the AOAC Official
Method 2007.01 and the EN 15662 method,. Taking into account
the association that takes place between the planar hydrophobic
chemical structure of PAHs and fatty components of the biological
matrices, and considering the good recovery percentages ob-
tained, they suggested that this mixture of solvents leads to more
intimate interaction with fatty fish matrices, and that the misci-
bility of acetone and EtOAc with water allows recovery of PAHs
trapped in water-sealed matrix pores, making them available for
their transfer to isooctane. It is also worth mentioning that, due to
the different natures of the analyzed samples, on some occasions,
Table 4
Some examples of the application of the QuEChERS method to the analysis of PAHs

Analytes Sample Extraction Sorbents in the d-SPE step Analytical Recovery (%) LODs Comments Ref.
(amount) technique
Solvents Salts

16 PAHs Kindling-free- 10 mL ACN 6 g MgSO4, 1.5 g NaOAc 400 mg PSA, 1200 mg MgSO4, GC-MS 71–104 0.1–2 μg/Lb 10 mL of water were added [78]
charcoal grilled 400 mg C18 silica gel particles initially; commercial
poultry meat, red QuEChERS kits
meat and seafood

M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185

(5 g)
16 PAHs Wild and 10 mL ACN 4 g MgSO4, 1 g NaCl 900 mg MgSO4, 150 mg PSA GC-MS/MS 89–112 0.01–0.99 μg/L 5 deuterated ISs [79]
commercial
mussels (10 g)
16 PAHs Rice grain (10 g) 10 mL ACN 1% 6 g MgSO4, 1.5 g NaOAc 150 mg MgSO4, 50 mg PSA GC-MS 70–122 - 10 mL of water were added [80]
HOAc (v/v) initially; 6 deuterated ISs
b
16 PAHs Meat (5 g) 10 mL ACN 6 g MgSO4, 1.5 g NaOAc 400 mg PSA, 1200 mg MgSO4, GC-MS 71–104 0.1–2 μg/L 10 mL of water were added [81]
400 mg C18 initially; commercial
QuEChERS kits
16 PAHs Oysters (10 g) 15 mL ACN 6 g MgSO4, 1.5 g NaCl 50 mg PSA, 150 mg MgSO4 UHPLC-MS 71–110 13–129 μg/kg 7 mL of water were added [82]
initially; commercial
QuEChERS kits
16 PAHs Chicken and duck 10 mL ACN 6 g MgSO4, 1.5 g NaOAc 400 mg PSA, 1200 mg MgSO4, GC-MS 71–104 0.1–2 μg/Lb 10 mL of water were added [83]
meat (5 g) 400 mg C18 initially; commercial
QuEChERS kits
16 PAHs Shrimp (10 g) 10 mL ACN 6 g MgSO4, 1.5 g NaCl 50 mg PSA, 150 mg MgSO4 LC-MS/MS 70–120a 20–510 μg/kg Commercial QuEChERS kits [84]
12 PAHs Ham (8 g) 10 mL EtOAc 4 g MgSO4, 1 g NaCl 0.150 g PSA, 0.300 g C18, GC-MS 72–111 0.1–1 μg/kg Anthracene d10 as IS [85]
0.900 g MgSO4
14 PAHs Manila clams, hard 5 mL ACN 2 g MgSO4, 0.5 g NaCl 50 mg PSA, 150 mg MgSO4 HPLC-FD 87–116 0.0500–0.5270 μg/kg Extraction step was carried out [86]
clams, oysters and twice
cockles (2 g)
12 PAHs Tea (1 g) 10 mL ACN 4 g MgSO4, 1 g NaCl 0.15 PSA, 0.15 g SAX, 0.9 g GC-MS 50–120 - 10 mL boiling water were [87]
MgSO4 added initially; 1 deuterated
IS; LLE step after QuEChERS
17 PAHs Sea urchin (1 g) 1 mL ACN 600 μg MgSO4, 150 μg NaOAc 90 mg MgSO4, 30 mg PSA, GC-MS/MS 70–120a 0.7–1.5 μg/kgb - [88]
15 mg C18
33 PAHs Salmon (1 g) 2 mL acetone: 6 g MgSO4, 1.5 g NaOAc; 4 g 50 mg PSA, 50 mg C18, 150 mg GC-MS 70–120a 2–10 μg/kg 2 deuterated ISs; commercial [89]
EtOAc:isooctane MgSO4, 1 g NaAc, 1 g tris-Na, MgSO4 QuEChERS kits
(2:2:1, v/v/v) 0.5 g di-Na
16 PAHs Fish (5 g) 8 mL ACN 6 g MgSO4, 1.5 g NaOAc 900 mg MgSO4, 300 mg PSA, LC-FD 70–120a 0.09–1.40 μg/L Commercial QuEChERS kits [90]
150 C18
5 OH-PAHs Milk (1200 μL) 300 μL ACN 480 mg MgSO4, 120 mg NaCl 30 mg PSA, 30 mg C18 CE-UV 80–105 0.98–3.72 μg/kg Hydrolysis step prior to [91]
QuEChERS
a Recovery values were found at 70–120% for most of analytes determined.
b
Instrumental LOD.

179
180 M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185
the addition of a certain amount of water has also been necessary
to enable the extraction process [78,80–83,87].
From review of the literature, it is also clear that the sorbent most
commonly used for the d-SPE step has been PSA [79,80,82,84,86],
in most cases with MgSO 4 , and also in combination with C 18
[78,81,83,85,88–91]. However, Sadowska-Rociek et al. [87] sug-
gested an interesting alternative that involved adding PSA and SAX
(a strong ion exchanger) after the study of several sorbent combi-
nations (PSA, PSA + C18, PSA + GCB, PSA + NH2). The fact that PAHs
are apolar planar compounds has precluded the application of GCB
for clean-up purposes, which is well known to retain planar com-
pounds. In fact, few articles have tested this sorbent for the d-SPE
procedure [87].
As well known, PAHs can be analyzed well by GC or LC, which
have therefore also been used after the QuEChERS extraction pro-
cedure, especially GC. In general, GC [78–81,83,85,87–89] and LC
[82,84] {also in its UHPLC format [82]} have been coupled to an MS
detection system, though, for LC, FD was also found appropriate due
to its high selectivity and sensitivity [86,90].
In general, all these methods developed for the analysis of PAHs,
regardless of the separation technique used for their determina-
tion, have provided recovery percentages of 70–120%, except for few
analytes. However, we should take into account that many of the
works included in Table 4 used deuterated ISs [79,80,85,87,89].
Finally, it is important to note that most of the methodologies de-
veloped provided LODs of a few μg/kg or μg/L.
4.5. Miscellaneous
Apart from pesticides, pharmaceuticals, mycotoxins and PAHs,
other families of compounds have also been determined using
QuEChERS as the extraction method. Some significant examples are
shown in Table 5 {e.g., macrocyclic lactones [92], lipids [93], anti-
oxidants and food additives [94], hormones [95], UV filters [95],
endocrine disruptors [95–97], PCBs [32], flame retardants [32], PBDEs
[32], perfluoroalkyl substances [98,102], phthalates [99], isoflavones
[103] surfactants [104] and acrylamide [100,105]}. Such analytes have
been determined in environmental matrices {including water [95],
soils and sediments [95]}, foods {i.e., milk and its derivatives
[92,94,98,106,107], fish and seafood [31,32,96,98], meat [98,106],
honey and beehive products [97], and fruits, vegetables and de-
rivatives [99,107,108]} and biological matrices {i.e., blood [101,109],
urine [93,101], plasma [93], cerebrospinal fluid [101], stomach
content [101] and human organs [101]}.
In a good number of works, as described above, conditions af-
fecting extraction efficiency (i.e., type and volume of solvent of
extraction, sample pH, type and amount of salt to induce the salting-
out effect, and type and amount of sorbent in d-SPE clean-up) were
mainly optimized by a step-by-step approach, though, in some cases,
experimental designs were used [94].
Regarding the extraction step, ACN is again the preferred solvent
in the majority of papers for miscellaneous compounds, but other
solvents (e.g., EtOAc) or mixtures (e.g., CHCl3:MeOH) have been used
because they provided better results [93]. Similarly, the chosen salt
has also been NaCl. Regarding sample pH, authors have mostly se-
lected the citrate-buffered method [103], the acetate-buffered
method (using acetate, almost exclusively NaOAc and HOAc)
[93–95,101], formic acid [98], or non-buffered methodologies
[32,92,96,97,99,109].
Finally, regarding d-SPE sorbents, the most popular has been,
without doubt, PSA. Moreover, PSA has been very frequently
combined with other materials {e.g., GCB [95,96,98], C 18
[93,94,96,98,101,109] or Z-Sep [32]}. It is important to note that, in
a good number of cases, especially those methodologies that have
analyzed biological matrices [93,101,109], the sample amount has
been dramatically reduced (and therefore the other reagents have
been properly scaled down) because of its low availability (typical
sample amounts for these matrices have been 100–1000 μL, or ~1 g).
Apart from the comparison between acetate-buffered and citrate-
buffered procedures, which has been common in some of these
works [110–113], different authors have compared the efficiency,
The speed and the ease of QuEChERS methods with other well-
established procedures [LLE, the Folch method (used in the extraction
of lipids), pseudo-modified QuEChERS (avoiding the d-SPE step),
microwave-assisted solvent extraction, classical SPE, pressurized
liquid extraction (coupled to gel-permeation chromatography or
SPE)] for the extraction of miscellaneous compounds [93,97,114].
Although QuEChERS provided better (or comparable) results in the
majority of the manuscripts (in terms of not only efficiency and
speed but also solvent and reagent consumption and economic cost),
there are examples in which QuEChERS methods has been less ef-
ficient [97,108]. As an example, Domínguez-Álvarez et al. [97]
determined five endocrine disruptors in honey by CE-MS using
QuEChERS (extraction with 10 mL of ACN, 6 g MgSO4 and 1.5 g NaCl
of 10 g of honey diluted with 5 g of water, and clean-up with 150 mg
MgSO4 and 50 mg PSA) and two different LLE methods (LLE 1: ex-
traction with 9 mL of n-hexane of 25 g of honey diluted with 10 g
of water, LLE 2: extraction similar to LLE 1, with a shaking step after
centrifugation and then repetition of the centrifugation step). All
the analytical parameters obtained with QuEChERS were worse, in-
cluding matrix effect (95–124% for QuEChERS, 80–120% for LLE 1
and 70–100% for LLE 2), extraction recovery (≤3% for QuEChERS,
1–10% for LLE 1 and 25–70% for LLE 2) and process efficiency (≤3%
for QuEChERS, 0.3–11% for LLE 1 and 18–65% for LLE 2). The reason
might have been the extremely high sugar content of these samples.
In that case, it would have been particularly interesting to study in
depth what would happen with suitable modifications to the
QuEChERS method.
Although the QuEChERS method generally provided suffi-
ciently clean samples, on certain occasions (due the nature of the
sample or analytes), an additional extraction step was found nec-
essary (principally DLLME or SPE). For example, in Cunha et al. [96],
BPA and BPB were determined in canned tuna, sardines, macker-
els, squid, octopus, mussel, eel, anchovy and codfish. After the
QuEChERS methodology (using 5 mL of n-heptane to eliminate lipids
of 10 g of sample, later extracted with 10 mL of ACN, 4 g of MgSO4
and 1 g of NaCl and cleaned with 1.2 g of MgSO4, 120 mg C18 and
50 mg of GCB), a DLLME procedure [using 1 mL of ACN extract as
dispersant solvent with 5% (v/v) K2CO3 solution to regulate the acidity,
50 μL of tetrachloroethylene as extraction solvent with 150 μL of
acetic anhydride as derivatizing agent and 4 mL of water] was de-
veloped for further clean-up and concentration. The established
method (with LODs of 0.2–0.4 μg/kg) was then utilized for screen-
ing for the presence of BPA and BPB in canned seafood from Portugal,
detecting BPA in 83% of samples at concentrations of 1.0–99.9 μg/
kg and BPB in one sample (21.67 μg/kg).
It is worth mentioning that the high efficiency of the QuEChERS
method has favored the development of numerous procedures for
the simultaneous determination of diverse compounds with dif-
ferent properties (including pesticides, pharmaceuticals, PAHs,
personal-care products, PCBs, hormones, and PBDEs) [32,95] in foods
(including animal tissues, fruits and vegetables) and environmen-
tal matrices. The positive results obtained in this type of application
[32,95] demonstrate the potential of QuEChERS as a general, non-
selective procedure, allowing the simultaneous extraction of different
kinds of compound with very dissimilar properties. One example
is the work of Fan et al. [108], in which 201 diverse chemical pol-
lutants were analyzed in tea by GC-MS or GC-MS/MS. In this work,
three different extraction methods were compared:
• a double acidic ACN extraction followed by clean-up by classi-
cal SPE;
Table 5
Examples of the application of the QuEChERS method to the analysis of miscellaneous compounds

Analytes Sample Extraction Sorbents in Analytical Recovery LODs Comments Ref.

(amount) the d-SPE step technique (%)
Solvents Salts

59 multi-residue compounds (42 Shrimp (10 g) 10 mL ACN 5 g HCO2NH4 75 mg MgSO4, 25 mg LPGC-MS/MS 70–120a <5 μg/kg 2 deuterated ISs [24]
pesticides, 5 flame retardants, PSA, 25 mg C18, < 0.5 μg/kg for PCBs
9 PCBs and 3 PAHs) 25 mg
Z-Sep
68 multi-residue compounds (13 Catfish (10 g) 10 mL ACN 4 g MgSO4, 1 g NaCl 150 mg MgSO4, LPGC-MS/MS 60–124 0.1–10 μg/kgb 12 isotopically labeled ISs [32]
flame retardants, 18 pesticides, 50 mg Z-Sep
14 PCBs, 16 PAHs and 7 PBDE)
5 macrocyclic lactones Milk (10 mL) and 10 mL ACN 4 g MgSO4, 1 g NaCl 300 mg MgSO4, HPLC-FD 83–112 Milk: 0.4–3.2 μg/L - [92]
Yogurt (10 g) 100 mg PSA Yogurt: 0.6–0.9 μg/
kg
19 lipids Plasma (100 μL) and Plasma: 200 μL Plasma: 125 mg 50 mg C18 UHPLC-MS/MS Plasma: 81–100 - Efficiency compared with Folch [93]
urine (1 mL) CHCl3:MeOH 2:1 MgSO4, 25 mg Urine: 79–99 method (similar results);13:0/
(v/v) CHCl3:MeOH NaOAc 13:0-phosphatidylcholine and

M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185

(2:1, v/v) Urine: 1.25 g MgSO4, 15:0/15:0-phosphatidylglycerol
Urine: 2 mL 0.25 g NaOAc as IS
CHCl3:MeOH
(2:1, v/v)
43 antioxidants, preservatives Milk, milk beverages 10 mL ACN 1% HOAc 6 g MgSO4, 1.52 g 1.2 g MgSO4, 410 mg UHPLC-MS Milk: 89–108 0.0001–3.6 μg/kgc A full factorial central composite [94]
and synthetic sweeteners and yogurt (15 g) (v/v) NaOAc PSA, 404 mg C18 design was used for optimization
of factors
20 multi-residue compounds (2 Watercourse, lake 10 mL ACN 6 g MgSO4, 1.5 g PSA and GCB LC-MS/MS and GC-MS >72 0.5–23.0 μg/kgd Addition of water to samples; [95]
alkylphenols, 3 PAHs, 6 and coastal NaOAc Pentafluorobenzyl bromide as IS;
pesticides, 5 pharmaceutical sediments (2 g) commercial QuEChERS kits
residues, 2 hormones, 1 UV
filter and BPA)
BPA and BPB Canned seafood 10 mL ACN 4g MgSO4, 1 g NaCl 1.2 g MgSO4, 120 mg GC-MS 68–104 0.2–0.4 μg/kg Addition of 10 mL of water to [96]
(tuna, sardines, C18, 50 mg GCB samples; application of DLLME as
mackerels, squid, a second extraction procedure;
octopus, mussel, eel, d16-BPA as IS
anchovy and
codfish) (10 g)
5 endocrine disruptors Honey (10 g) 10 mL of ACN 6 g MgSO4, 1.5 g 150 mg MgSO4, CE-MS ≤3 - Addition of 5 g of water to [97]
NaCl 50 mg PSA samples; method compared with
two different LLE procedures; ;
2,4-dichlorophenol as IS;
commercial QuEChERS kits
21 perfluoroalkyl substances Butter (5 g) Butter: 20 mL ACN, 6 g MgSO4, 1.5 g Butter: 3.6 g MgSO4, UHPLC-MS/MS 73–128 Butter: 2.5–125 ng/ Addition of 10 mL of water to [98]
Cheese, hen eggs, 0.5 mL formic acid NaCl 360 mg C18, 180 mg kg samples (except to butter); 9
pork, canned pork, Other samples: GCB Other samples: isotopically labeled ISs
beef, poultry, pig 15 mL ACN, 0.2 mL Other samples: 1.8 g solid: 1–10 ng/kg;
liver, bovine liver, formic acid MgSO4, 180 mg C18, liquid: 1–5 ng/L
rabbit, sheep, lamb, 90 mg GCB
farmed freshwater,
marine fish, seafood,
and whole and
skimmed cow milk
(7.5 g)
5 phthalates Fruit jelly (10 g) 10 mL ACN 4 g MgSO4, 1 g NaCl 200 mg MgSO4, LC-MS/MS 84–104 0.09–3.68 μg/L - [99]
25 mg PSA
Acrylamide Fried potatoes, 10 mL ACN 5 g MgSO4, 1 g NaCl 150 mg Al2O3 LC-MS 90–97 1.50–4.04 μg/kg Initial defatting process with [100]
eggplant and baked n-hexane
Sudanese foods (1 g)
α-pyrrolidinobutiophenone Blood, urine, 1 mL ACN Fluids: - 150 mg MgSO4, UHPLC-MS/MS 83–103 Fluids: 0.05 ng/L Use of standard addition [101]
stomach content Tissues: 400 mg 25 mg PSA, 25 mg Tissues: 0.1 μg/kg method;
and cerebrospinal MgSO4, 100 mg C18 α-pyrrolidinovalerophenone as
fluid (100 μL) NaOAc IS; commercial QuEChERS kits
Lung, liver, kidney,
pancreas and spleen
(1 g)
a
Recoveries values were found at 70–120% for most of analytes determined.
b
Lowest calibration level (LCL).
c Decision limit (CCα).

181
d Limits of quantification (LOQ).
182 M.Á. González-Curbelo et al./Trends in Analytical Chemistry 71 (2015) 169–185
• the acetate-buffered approach; and,
• a double extraction with ACN with NaCl, removing of water with
MgSO4 and clean-up by the previously selected classical SPE
cartridge.
Although the QuEChERS method was easy to operate, the first
method showed superior efficiency in removing pigments and ex-
tracted 92.0% of compounds with recoveries of 70–110% while the
QuEChERS method recovered 91.8% with the recoveries in the same
range. The third method extracted no more than 4% of analytes with
high recoveries.
Finally, as also happened in previous applications, in the vast ma-
jority of papers, the separation techniques most commonly used
were LC {both classical LC [95,99,104,109] and UHPLC
[93,94,98,101,106]} coupled to MS [94,104,106] or MS/MS
[93,95,98,99,101,109] and GC to MS [31,95,96,108] or MS/MS [32,108].
Less common has been the application to other detection systems
[92]. In a good number of these works, a high number of analytes
(sometimes even higher than one hundred [106,108]) has been de-
termined in different matrices with acceptable results in terms of
accuracy and reproducibility.
Regarding the application of CE, some time-saving applica-
tions were proposed [97,103].
5. Conclusions
The popularity of the QuEChERS method has skyrocketed in
recent years in a way that no one could imagine. The main reason
has been that it has addressed the needs of modern laboratories,
including reduction in the use of solvents and materials, and de-
creased the time and the cost of analysis. QuEChERS is undoubtedly
the most frequently used pretreatment technique in residue labo-
ratories everywhere for pesticides analysis in foods, but it has also
proved itself to be capable of adaptation to new challenges, showing
its usefulness in coping with a wide variety of other analytes in a
wide diversity of commodities. Even so, new QuEChERS concepts
and modifications are welcome.
The QuEChERS method has really managed to increase sample
throughput while maintaining good results, which is what was being
sought in routine analysis. Despite the ease and the speed of
QuEChERS, the method is still a manual procedure and the possi-
bility of automation has been considered to minimize sample
manipulation and to increase analytical throughput. However, in
practice, it has not gone beyond the use of shakers and commer-
cial kits to facilitate the procedure and to reduce operator fatigue.
The number of published articles in which the QuEChERS method
has been used is really high and has greatly increased since its in-
troduction in 2003. However, a good number of works claimed to
use it when only one of the two steps of the method was applied.
Most of these works, regardless the types of analyte or matrix used,
directly applied previously published conditions. In these cases, par-
ticularly when analytes other than pesticides (as well as new
matrices) were selected, it was quite difficult to evaluate if certain
parts of the method were really necessary for the final purpose. As
a result, in certain cases, it was desirable to try to demonstrate fully
the suitability of the method, as previously for pesticide analysis.
Considering the separation methods that have been used,
QuEChERS has been widely combined with different LC and GC in-
struments because of their well-established performance in analytical
laboratories, private and public. However, despite CE not being a
routine analytical technique for many of the target analytes that have
been extracted with the method, for some applications, authors have
also tried to combine QuEChERS with CE, resulting in acceptable
and feasible approaches. However, the number of published appli-
cations is very limited (compared with the use of LC or GC) because,
in CE, the final extracts need to be sufficiently clean and with low
conductivity to avoid current breakdowns. These essential require-
ments are not easily fulfilled in QuEChERS, due the use of different
salts that can affect the ionic strength of extracts, so explaining the
scarcity of papers.
Since the first work, new sorbents have also been proposed and
tested for the d-SPE step. In this sense, and taking into account
current research in the field, we expect in the near future the in-
troduction of new sorbents capable of providing a suitable
elimination of the matrix, while maintaining a high recovery of the
analytes. In all probability, this aspect, together with new at-
tempts to try to automate the procedure, will be the next steps in
the evolution of the QuEChERS method, which seems to be quite
difficult to displace at the moment.
Acknowledgements
B.S.R. thanks the Local Government of the Canary Islands for the
FPI grant at the University of La Laguna. A.V.H.H. wishes to thank
CajaSiete for his grant (2014 proposal) in the Servicios Generales de
Apoyo a la Investigación (SEGAI).
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