Talanta 71 (2007) 503–514

Review

Chromatographic-based methods for pesticide determination

in honey: An overview
R. Rial-Otero, E.M. Gaspar, I. Moura, J.L. Capelo ∗
REQUIMTE, Departamento de Quı́mica, Faculdade de Ciências e Tecnologı́a, Universidade Nova de Lisboa,
2829-516 Monte de Caparica, Portugal
Received 16 December 2005; received in revised form 18 April 2006; accepted 12 May 2006
Available online 21 June 2006

Abstract
Nowadays the control of pesticides in honey is an issue of primary health importance as consequence of the increasing content of these
chemicals in the aforementioned matrix. This poisoning has led to the worldwide increasing loss of bees since 1995. From Europe to Canada,
scientist, beekeepers and chemical companies disagree about the reasons that have led to colony losses higher than 50% in some areas. This problem
has become a public health issue due to the high honey worldwide consumption. The presence of pesticides in honey has been directly related
to bees’ mortality by some researchers through pesticide presence in (1) pollen, (2) honeycomb walls, (3) own bees and (4) honey. In this work
we describe the actual state-of-the-art for pesticides determination in honey along with a review in this subject focused on sample treatments and
instrumentation. Finally, future trends are also commented.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Pesticide; Honey; Sample treatment; GC; HPLC

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
2. Stability of pesticides and standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
3. Sample treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
3.1. SE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
3.2. SFE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506
3.3. SPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
3.4. MSPD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
3.5. SPME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
3.6. SBSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
4. Chromatographic separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
5. Pesticide levels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
6. Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512

Abbreviations: AED, atomic emission detector; APcI, atmospheric pressure chemical ionization; API, atmospheric pressure ionization; DAD, diode array
detector; EC, amperometric detector; ECD, electron capture detector; EPA, Environmental Protection Agency; ESI, electrospray ionization; EU, European Union; FID,
flame ionization detector; FLD, fluorescence detector; FPD, flame photometric detector; GC, gas chromatography; HPLC, high performance liquid chromatography;
LOQ, limit of quantification; LOD, limit of detection; MRLs, maximum residual limits; MSD, mass spectrometry detector; MSPD, matrix solid-phase dispersion;
NPD, nitrogen–phosphorus detector; OCPs, organochlorine pesticides; ODS, octadecylsilane; ONPs, organonitrogen pesticides; OPPs, organophosphorus pesticides;
PDMS, polydimethylsiloxane; PFPD, pulsed flame photometric detector; R.S.D., relative standard deviation; SBSE, stir bar sorptive extraction; SE, solvent extraction;
SFE, supercritical fluid extraction; SPE, solid-phase extraction; SPME, solid-phase microextraction; TSD, thermoionic-specific detector; UV, ultraviolet detector
∗ Corresponding author. Tel.: +351 21 294 9649; fax: +351 21 294 8550.

E-mail address: jlcapelom@dq.fct.unl.pt (J.L. Capelo).

0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2006.05.033
504 R. Rial-Otero et al. / Talanta 71 (2007) 503–514

7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513

1. Introduction frequently substances used to control varroatosis and ascosphe-

riosis are the acaricides amitraz, cymiazole, bromopropylate,
In the long term, the use for agriculture of chemical com- coumaphos, flumethrin, and fluvalinate (see Table 1). The use
pounds such as insecticides, fungicides, herbicides, organophos- of these compounds inside beehives implies a risk of direct con-
phorus pesticides (OPPs) and organochlorine pesticides (OCPs), tamination of honey and other hive products [5,6]. The ultimate
can cause the contamination of the environment. In some cases, consequence of this intensive use of pesticides is the total colony
the chemicals utilized to control plagues remains in the plant in collapse, as it has been reported for some geographical areas [7].
which they were spread, affecting other insects different from Since 1998 European and Canadian beekeepers have reported
the ones against the chemicals were developed for. Thus, dif- serious losses of honeybees that they have attributed to the use of
ferent pesticides can be introduced in the food chain by the the pesticides imidachloprid and fipronil in the agriculture prac-
bees through honey, affecting human health, when they come in tices.In addition to the important problem for public health, the
contact with nectar and pollen from blossoms in lands where pre- presence of the aforementioned substances in honey decreases
viously was used a pesticide, for instance, for beat control [1,2]. its quality. According to the European Union (EU) regulations,
Furthermore, diseases affecting bee’s larvae, namely (i) Amer- honey as a natural product must be free of chemicals [8]. As
ican foulbrood (Paenibacillus larvae), (ii) European foulbrood showed in Table 1, different national regulations have estab-
(Melissococcus plutonius), and (iii) Chalkbrood (Ascosphaera lished maximum residual levels (MRLs) of pesticide in honey.
apis) or parasite infestation such as (1) Nosema (Nosema apis), The Council Regulation 2377/90/EEC of EU and their subse-
(2) amoeba (Malpighamoeba mellifica), (3) varroa mites (Var- quent modifications has established MRLs in honey of three
roa Jacobsoni), and (4) tracheal mites (Acarapis woodi) [3] has acaricides namely, amitraz, coumaphos and cymiazole, of about
forded beekeepers to use chemical therapeutic treatments inside 0.2, 0.1 and 1 mg kg−1 , respectively [9]. On the other hand,
beehives [4]. The two principal routes of honey contamination the US Environmental Protection Agency (EPA) has established
with pesticides are shown in Fig. 1. As an example, the most MRLs for the same compounds as follows: amitraz, coumaphos,

Fig. 1. Ways of honey contamination with pesticides.

 R. Rial-Otero et al. / Talanta 71 (2007) 503–514 505

Table 1
Toxicity categories, acute LD50 in rats, and maximum regulated residual limits in frequently used substances for bee diseases control
Insecticide Toxicity Acute LD50 values (mg/kg) Maximum residual limits (MLRs) in honey (mg/kg)
category
Oral Dermal EUa USAb Germanyc Switzerlandc Italyc Netherlandsc

Amitrazd III 523–800 >1600 0.2 1 0.01 0.01 0.01 0.02

Coumaphos II 13–41 860 0.1 0.1 n.f. 0.01 n.f. 0.05
Cymiazole 725 >3100 1 e 0.01 n.f. 0.01 n.f.
Bromopropylate 2784–3880 >10000 e e 1 1 0.01 n.f.
Flumetrin 258 >150 e e 0.01 0.05 0.01 n.f.
Fluvalinate II 261–282 >20000 e 0.05 0.01 n.f. 0.01 0.05
Imidachloprid II–III 450 >5000 n.f.e n.f. n.f. n.f. n.f. n.f.
Fipronil II 296 374 n.f.e n.f. n.f. n.f. n.f. n.f.

n.f.: not found.

a Council regulation No 2377/90/EEC and their subsequent modifications.
b Food and Drug Administration of the United States. Pesticides tolerances (http://www.cfsan.fda.gov 2003).
c A review of treatment options for control of varroa mite in New Zealand. Report to the Ministry of Agriculture and Forestry (http://www.biosecurity.govt.nz,

2005).
d Sum of amitraz and all metabolites containing the 2,4-dimethylaniline moiety, expressed as amitraz.
e Do not have a MRL.

and fluvalinate of 1, 0.1 and 0.05, respectively [10]. To assess
pesticide residue levels in honey and their compliance with qual-
ity standards fixed by the UE and/or national regulations, several
methods have been developed.
The aim of this work is to critically review the literature meth-
ods for pesticide analysis in honey, including critical aspects
such as sample preparation and matrix effects.
2. Stability of pesticides and standards
The pH of honey is acid, between 3.2 and 4.5, and in these
conditions few pesticides are naturally degraded [11]. Let see
two examples. Firstly, amitraz degradation in honey was virtu-
ally complete and no amitraz traces were observed after 3, 15
and 30 days, for a preservation temperature of 40, 20 and 4 ◦ C,
respectively [11]. Secondly, the chlordimeform content varied
from 2 to 0.15 mg kg−1 after 28 weeks [12]. This is important
because suggests that for some pesticides the analysis should be
performed as soon as possible, avoiding long periods of sample
storage. In addition, the analytical community should be con-
cerned with the toxicity of the degradation compounds of these
pesticides. To the best of our knowledge, little has been done
to this respect. On the contrary, other pesticides can remain
unaltered for a long period of time. Thus, tau-fluvalinate is a
pesticide with a high persistence in honey and its level did not
decrease with time, even during the honey blending process
or after storage at 35 ◦ C in the dark for 8 months [13]. Stor-
age conditions can also affect pesticide stability. For instance,
exposure to light can decompose some pesticides, such as thy-
mol and rotenone [11]. For this reason pesticide standards and
honey samples must be stored in dark bottles. Another important
issue in pesticide determination is the solvent used for standard
preparation, which must be carefully chosen or at least tested
for pesticide stability. As an example, rotenone is unstable in
methanol and degradations rates of 50%, 30%, 10%, and 1%
have been observed for standard concentrations of 0.1, 1, 10, and
100 mg l−1 , respectively, after a 2 months period of storage at
4 ◦ C [14].
3. Sample treatment
Since honey is a difficult matrix, sample pre-treatment is
essential in order to obtain reliable results. During the last
decades, different methodologies have been proposed with the
aims of (i) to diminish sample handling and (ii) to reduce toxic
wastes [2]. The extraction of pesticides from honey is usually
done by one of the following procedures: (i) solvent extrac-
tion, SE; (ii) supercritical fluid extraction, SFE; (iii) solid-phase
extraction, SPE; (iv) matrix solid-phase dispersion, MSPD; (v)
solid-phase microextraction, SPME, and (vi) stir bar sorptive
extraction, SBSE. Those sample preparation procedures are
depicted in Fig. 2 and their main parameters are summarized
in Table 2.
3.1. SE
In the SE extraction procedure we may distinguish three steps.
In the first one, honey is diluted in water [12,15–20] or water
mixtures such as acetone–water [21] or methanol–water [22] in
order to obtain a better sample homogenization prior to analyte
extraction. In the second step the pesticides are extracted with
different water-immiscible solvents according to the pesticide’s
polarity. Different extracting solvents or solvent mixtures can
be found in literature such as dichloromethane [11,19,21], ethyl
acetate [20,22,23], or mixtures of benzene–isopropanol [16],
hexane–acetone [12,17,18], hexane–glacial acetic acid [15] and
hexane/2-propanol [11]. The polarity of the extracting solvent
is a trade-off between an acceptable recovery and a good mea-
surement. For instance, the use of acetone in combination with
hexane increases the solvent polarity and the recovery of the
polar analytes, but the number and concentration of non-desired
co-extracted substances is also increased and a worst stability
of the baseline is obtained [17,18]. In some cases, acidifica-
tion can helps to increase analyte extraction, as for fluvalinate.
For the latter compound the use of the aqueous phase acidified
with glacial acetic acid decreased the co-extraction of interfer-
ing compounds, being the recovery of fluvalinate into hexane
506 R. Rial-Otero et al. / Talanta 71 (2007) 503–514

Fig. 2. Sample preparation techniques for the determination of pesticides in honey. (i) SE, solvent extraction; (ii) SFE, supercritical fluid extraction; (iii) SPE,
solid-phase extraction; (iv) SPME, solid-phase microextraction; (v) SBSE, stir bar sorptive extraction.

facilitated [15]. Moreover, acidification of the sample seems
to improve the analytical performance in the case of benomyl
and carbendazim [23]. On the other hand, increased yields of
extraction are obtained when sonication with bath is used in
order to speed up the SE procedure [1,14]. Finally, in the third
step, the extract obtained after sample treatment is cleaned (i)
to avoid non-desired co-extracted high molecular weight com-
pounds that can contaminate the chromatographic system and
(ii) to diminish interfering compounds that can make the chro-
matogram difficult to understand, mainly due to the presence
of overlapping peaks. To achieve the latter objective, different
strategies has been proposed such as liquid–liquid partitioning
[11], on column octadecylsilane (ODS) cleaning [17,18,22] and
gel permeation chromatography [19,21]. The main drawbacks
of the solvent extraction approach are the following: (1) large
amounts of solvents are used, (2) is a time-consuming approach,
(3) intense sample handling is required, and (4) large amounts
of waste are generated.
3.2. SFE
SFE has shown to be an efficient and rapid method for the
isolation of pesticides from honey [2,4,24]. This procedure uti-
lizes the unique properties of supercritical fluids to facilitate
the extraction of pesticides from solid samples. A supercritical
fluid is a substance above its critical temperature and pressure.
Carbon dioxide (CO2 ) has become the solvent of choice for
most SFE applications. CO2 has a low supercritical temper-
ature (31 ◦ C) and pressure (73 atm), and is available at high
purity. Supercritical CO2 is a good solvent for the extraction
of non-polar or moderately polar compounds. The extraction
efficiency of polar compounds by CO2 can be improved by the
addition of small quantities of polar organic solvents used as
modifiers [25]. SFE has gained increased attention as a poten-
tial alternative for conventional SE due to its properties: (i) it
is fast (10–60 min), (ii) it uses minimum amount of solvents
(5–10 ml), (iii) CO2 is non-toxic, non-flammable and environ-
mentally friendly, (iv) selective extraction without additional
clean-up [26] can be done, and (v) little sample amounts (<10 g)
[27,28] of sample are required. Thus, Rissato et al. [2] have
shown that better precision and higher recovery yields were
obtained for 32 pesticides with SFE (OPPs, OCPs, ONPs, and
pyretroids) than with traditional SE, while detection limits were
similar for both methodologies. However, one of the main limi-
tations of SFE is that can no be used to extract pesticides directly
from water due to the low solubility of CO2 in water. In these
 R. Rial-Otero et al. / Talanta 71 (2007) 503–514
Table 2
Main parameters of methodologies reported in literature for the extraction of pesticides from honey
Analytes/extraction procedure
Trichlorfon, dimethoate and pirimicarb. SE. 20 g of honey diluted with
20 ml of water was extracted with 100 ml of ethyl acetate in the presence
of 30 g sodium sulphate. Clean-up with gel permeation column S-X3
with ethyl acetate–cyclohexane (1:1) as mobile phase
Fluvalinate. SE. 5–10 g of honey diluted with 10–20 ml of methanol–water
(80:20, v/v) were extracted with 3 × 20 ml of ethyl acetate. Clean-up
with C18 cartridges and elution with 8 ml of n-hexane
Rotenone. SE. 10 g of honey were extracted with 2 × 30 ml of
dichloromethane for 2 min in an Ultra-Turrax blender
Fluvalinate, malathion and coumaphos. SE. 10 g of honey diluted with
20 ml of water were extracted with 2 × 10 ml of dichloromethane for
1 min. Clean-up with a mixture of silica gel with activated charcoal l
(15:1)
Fluvalinate SE. 10 g of honey diluted with 200 ml of water were extracted
with a mixture of 50 ml of n-hexane plus 25 ml of glacial acetic acid for
30 s and then extracted again with 2 × 50 ml of n-hexane
13 OCPs, 30 OPPs, five ONPs. SE. 15–20 g of honey diluted with 200 ml
of aceanol–water (1:1, v/v) were extracted with 10 ml of
dichloromethane, 3 ml of saturated NaCl solution and 50 ml of water and
then, extracted again with 2 × 10 ml of dichloromethane. Clean-up with
packed-column with 0.5 g of silica gel, 5 g of a mixture of activated
carbon and silica gel (1:15) and 1 g of Na2 SO4
Acrinathrin, 3-phenoxybenzaldehyde. SE. 1 g of honey diluted with 5 ml of
water were extracted with 3 × 25 ml of benzene–isopropanol (1:1, v/v)
for 20 min
Vinclozolin. SE. 1–5 g of honey diluted with 2 ml of water were extracted
with 2 × 30 ml of hexane–acetone (70:30, v/v) for 20 min. Clean-up with
C18 cartridge or florisil packed-columns
Chlordimeform, bromopropylate and amitraz. SE. 2 g of honey diluted
with 1 ml of water were extracted with 3 × 15 ml of n-hexane–acetone
(80:20, v/v) for 30 min. Clean-up with C18 cartridges
Chlordimeform and their metabolites 4-chloro-o-toluidine and
N-formyl-4-chloro-o-toluidine. SE. 1 g of honey diluted with 1 ml of
water at pH 9 and extracted with 2 × 25 ml of n-hexane–acetone (80:20,
v/v) for 30 min
Benomyl and carbendazim. SE. 1 g of honey were extracted first with 3 ml
of HCl (0.05 M) and 15 ml of ethyl acetate for 15 min, then with another
15 ml of ethyl acetate and finally with 3 ml of NaOH (0.1 M) plus 15 ml
of ethyl acetate
Coumaphos, bromopropylate, amitraz and fluvalinate. SE. 20 g of honey
were extracted twice with 60 ml of n-hexane, 30 ml of 2-propanol and
0.28% of ammonia for 2 min in an Ultra-Turrax blender. The combined
extracts were extracted with 50 ml of water with 0.28% ammonia and the
n-hexane phase was extracted twice with 50 ml of basic water (pH 10)
32 pesticides. SFE. 5 g of honey + 3 ml of water + 2 g of cellulose powder
were frozen and lyophilized. CO2 extraction with 10% of acetonitrile as
organic modifier. Conditions: 400 bar, extraction chamber Ta 90 ◦ C,
extraction time 20 min. Pesticides were collected on-line in a florisil
cartridge at 10 ◦ C and were eluted with 5 ml of
dichloromethane–n-hexane (80:20, v/v) and 5 ml of n-hexane–acetone
(60:40, v/v)
507
Technique/comments/quality parameters Ref.
GC–ECD (300 ◦ C), GC–TSD (300 ◦ C) or GC–PFPD (275 ◦ C). [20]
Columns: CP-Sil 8CB (25 m × 0.32 mm i.d., 0.25 ␮m), CP-Sil 19CB
(25 m × 0.32 mm i.d., 0.20 ␮m). Carrier gas: helium at a flow-rate of
3 ml min−1 . Recovery: 79–95%. R.S.D. < 17%
GC–ECD (280 ◦ C). Injector: PTV at 250 ◦ C. Column: SE-54 [22]
(12 m × 200 ␮m i.d.; 0.3 ␮m). GC–MSD electronic impact (ionization
potential 70 eV, transfer line 260 ◦ C). Injector: PTV at 250 ◦ C. Column:
SE-54 (12 m × 200 ␮m i.d.; 0.3 ␮m). Recovery: 93–100%.
R.S.D. < 8%. LOD: 0.008–0.018 mg kg−1
HPLC–DAD. Column: C18 (250 mm × 4.0 mm i.d., 5 ␮m). Mobile [11]
phase: acetonitrile–water (60:40, v/v) at a flow-rate of 1 ml min−1 .
Recovery: 91–110%. R.S.D. < 7%. LOD: 0.005 mg kg−1
Malathion and coumaphos GC–NPD (300 ◦ C). Injector: splitless at [19]
230 ◦ C. Column: HP-1 (15 m × 0.53 mm i.d.; 0.5 ␮m). Recovery:
79–95%. R.S.D. < 20%. LOQ <0.02 mg kg−1 . Fluvalinate. GC–ECD
(300 ◦ C). Injector: splitless at 250 ◦ C. Column: CP-Sil 8
(15 m × 0.53 mm i.d.; 1.5 ␮m). Recovery: 89–92%. R.S.D. < 7%. LOD:
0.005 mg kg−1
GC–ECD (300 ◦ C). Injector at 250 ◦ C. Column borosilicate glass [15]
column packed with 3% SP-2100 in isocratic mode. Recovery:
98–102%. R.S.D. < 10%. LOD: 3 mg kg−1
OCPs GC–ECD (280 ◦ C). Injector: splitless at 270 ◦ C. Column: [21]
Ultra-2 (25 m × 0.5 mm i.d.; 0.1 ␮m). Recovery: 87–105%. LOD:
<0.01 mg l−1 . 30 OPPs and five ONPs. GC–NPD (280 ◦ C). Injector:
split at 250 ◦ C. Column: BP-5 (25 m × 0.32 mm i.d.; 0.5 ␮m).
Recovery: 60–90%. LOD: <0.1 mg l−1 . Fenpropathrin, fluvalinate and
bromopropylate. GC–ECD (300 ◦ C). Injector: split at 250 ◦ C. Column:
BP-1 (25 m × 0.32 mm i.d.; 0.5 ␮m). Recovery: 79–85%. LOD:
<0.05 mg l−1
GC–FID (300 ◦ C). Injector: splitless at 225 ◦ C. Column: capillary [16]
column with 50% of phenyl methylpolysiloxane (30 m × 0.25 mm i.d.,
0.25 ␮m). Recovery: 91–97%. R.S.D. < 3.6%. LOD < 0.01 mg kg−1
GC–ECD (300 ◦ C). Injector: splitless at 200 ◦ C. Column: 007-17 [17]
(60 m × 0.25 mm i.d.; 0.25 ␮m). GC–MSD. Electronic impact
(ionization potential 70 eV, ion source 200 ◦ C, transfer line 280 ◦ C,
analyzer 100 ◦ C). Injector: splitless at 200 ◦ C. Column: DB-17
(30 m × 0.25 mm i.d.; 0.25 ␮m). Recovery: 98%. R.S.D. < 5%
GC–AED (280 ◦ C). Injector: splitless at 200 ◦ C. Column: DB-17 [18]
(30 m × 0.25 mm i.d.; 0.25 ␮m). GC–ECD–NPD (300 ◦ C). Injector:
splitless at 200 ◦ C. Column: DB-17 and -5 (30 m × 0.25 mm i.d.;
0.25 ␮m). Recovery: 91–97%. R.S.D. < 6%. LOD: <0.07 mg l−1
GC–MSD positive and negative chemical ionization with methane (ion [12]
source 200 ◦ C, transfer line 280 ◦ C, analyzer 100 ◦ C) or GC–AED
(280 ◦ C). Injector: splitless at 200 ◦ C. Column: DB-17
(30 m × 0.25 mm i.d.; 0.25 ␮m). R.S.D. < 18%
HPLC–FLD (λ excitation at 285 nm and emission at 317 nm). Column: [23]
C18 (150 mm × 3.9 mm i.d.) thermostated at 25 ◦ C. Mobile phase:
acetonitrile–water (40:60, v/v; pH 4) at a flow-rate of 1 ml min−1 .
Confirmation by HPLC–PB–MSD with C18 (250 mm × 2 mm i.d.)
column and using water–methanol (45:55, v/v) as mobile phase at
flow-rate of 0.3 ml min−1 . Recovery: 91–100%. R.S.D. < 5%
HPLC–DAD. Column: C18 (250 mm × 4.0 mm i.d., 5 ␮m). Mobile [11]
phase: acetonitrile–water (80:20, v/v) with 0.028% ammonia (pH 9) at
a flow-rate of 1 ml min−1 . Recovery: 86–103%. R.S.D. < 7%. LOD:
0.005 mg kg−1
GC–ECD (300 ◦ C). Injector: splitless at 250 ◦ C. Column: HP-608 [2]
(30 m × 0.25 mm i.d.; 0.25 ␮m). Confirmation by GC–MSD electronic
impact (ionization potential 70 eV; transfer line 280), equipped with a
LM-5 column (35 m × 0.25 mm i.d.; 0.25 ␮m). Recovery: 88–98%.
R.S.D. < 6%. LOD: 0.010 mg kg−1
508 R. Rial-Otero et al. / Talanta 71 (2007) 503–514
Table 2 (Continued )
Analytes/extraction procedure
Fluvalinate. SFE. 20 g of honey + 4 ml of water + 2 g of cellulose powder
were frozen and lyophilized. A fraction of 2 g of lyophilizate was
extracted using CO2 and 25 ␮l of benzene-isopropanol (7:3, v/v) as
organic modifier. Conditions: fluid density 0.45 g ml−1 , 138 bar,
extraction chamber Ta 70 ◦ C, flow-rate 0.8 ml min−1 , extraction time
20 min. Analyte was collected on a trap packed with stainless-steel balls
and was eluted at 75 ◦ C with 1 ml of methanol
Rotenone. Ultrasons. 2 g of honey diluted with 15 ml of water were
extracted with 2 × 15 ml n-hexane–dicholoromethane (50:50, v/v) with
ultrasonic bath for 20 min operated at 43 KHz
Atrazine and simazine Ultrasound. 5 g of honey were extracted with 20 ml
of benzene-water (1:1, v/v) for 3 × 20 min in an Ultrasonic bath (30 KHz
and 400 W)
Rotenone. SPE. 2 g of honey diluted with 25 ml of water were passed
through a C18 cartridge and the pesticide was eluted with 3 ml of
methanol
Vinclozolin. SPE. 1–5 g of honey diluted with 100 ml of water were passed
through a C18 cartridge and the pesticide was eluted with 2 ml of acetone
18 OCPs. SPE. 5 g of honey diluted with 25 ml of methanol and 2 l of water
at pH 2 were passed through a C18 cartridge and the pesticides were
eluted with 10 ml of hexane
4 acaricides. SPE. 5 g of honey diluted with 15 ml of methanol–water (1:1,
v/v) were passed through a C18 cartridge and the cartridge was washed
with 5 ml of methanol–water (1:1, v/v). Then, the pesticides were eluted
with 5 ml of hexane
Tau-fluvalinate. SPE. 5 g of honey diluted with 15 ml of ethanol–water
(1:1, v/v) were homogenized in an ultrasonic water bath and centrifuged.
The supernatant was passed through a C8 cartridge and the cartridge was
washed with 5 ml of ethanol–water (1:1, v/v) and 100 ␮l of hexane.
Pesticides were eluted with 2 × 2.5 ml of dichloromethane
9 acaricides. SPE. 0.5 g of honey diluted with 5 ml of aqueous buffer (1 M,
pH 6.0) were sonicated during 5 min. 4 ml of this solution were passed
through a C18 SPE cartridge. The cartridge was washed with 1 ml of
THF–aqueous buffer (1 M, pH 6.0) (10:90, v/v) and the pesticides were
eluted with 1 ml of THF
Amitraz, bromopropylate, coumaphos, cymiazole and fluvalinate. SPE.
10 g of honey diluted with 10 ml of water were passed through a system
of two C18 cartridges. The system was washed with 3 × 3 ml of water
and the pesticides were eluted with 5 ml of hexane–dichloromethane
(50:50, v/v)
22 OPPs. SPE. 5 g of honey diluted with 50 ml of water were passed
through a C18 packed-column. The pesticides were eluted with 10 ml of
ethyl acetate, 4 ml of methanol, and 1 ml of dichloromethane
42 carbamates, OCPs and OPPs. SPE. 5 g of honey diluted with 50 ml of
water were passed through a C18 packed-column. The pesticides were
eluted with 10 ml of ethyl acetate, 4 ml of methanol, and 1 ml of
dichloromethane
51 pesticides (insecticides, fungicides, acaricides and herbicides). SPE.
10 g of honey diluted with 10 ml de methanol–water (30:70, v/v) were
passed through a C18 packed-column. The column was washed with
5 ml of methanol–water (70:30, v/v) and the pesticides were eluted with
10 ml of hexane–ethyl acetate (50:50, v/v)
Technique/comments/quality parameters Ref.
HPLC–UV (254 nm). Column: C18 (150 mm × 4.6 mm i.d., 5 ␮m). [26]
Mobile phase: acetonitrile–water (80:20, v/v) containing 1.4% of
HAcO (0.01 M) at a flow-rate of 1.5 ml min−1 . Recovery: 53–94%.
R.S.D. < 3%. LOD: 0.02 mg kg−1
HPLC–UV at 210 nm. Column: C18 (250 mm × 4.6 mm i.d.). Mobile [14]
phase: acetonitrile–buffer phosphate (pH 7) (60:40, v/v) at a flow-rate
of 1 ml min−1 . Recovery: 59–102%. R.S.D. < 7%. LOD: 0.015 mg kg−1
TLC on HPTLC silica gel 60 F254 plates. Mobile phase: [1]
hexane–chloroformo–acetona (60:25:15, v/v/v). Recovery: 92–95%
HPLC–UV at 210 nm. Column: C18 (250 mm × 4.6 mm i.d.). Mobile [14]
phase: acetonitrile–buffer phosphate (pH 7) (60:40, v/v) at a flow-rate
of 1 ml min−1 . Recovery: 78–98%. R.S.D. < 7%. LOD: 0.015 mg kg−1
GC–ECD (300 ◦ C). Injector: splitless at 200 ◦ C. Column: 007–17 [17]
(60 m × 0.25 mm i.d.; 0.25 ␮m). GC–MSD. Electronic impact
(ionization potential 70 eV, ion source 200 ◦ C, transfer line 280 ◦ C,
analyzer 100 ◦ C). Injector: splitless at 200 ◦ C. Columns: DB17
(30 m × 0.25 mm i.d.; 0.25 ␮m). Recovery: 96–98%. R.S.D. < 4%
GC–ECD. Injector in splitless mode at 250 ◦ C. Column: a DB-1 [35]
(30 m × 0.32 mm i.d., 0.25 ␮m). Confirmation by GC–ECD equipped
with a DB-5 column (30 m × 0.25 mm i.d., 0.25 ␮m). Recovery:
48–125%. R.S.D. < 13%. LOD: 0.2 ␮g kg−1
HPLC–DAD. Column: ODS-2 (150 mm × 0.32 mm i.d., 5 ␮m). Mobile [37]
phase: methanol–water (90:10, v/v) at a flow-rate of 5 ␮l min−1 .
Recovery: 81–95%. R.S.D. < 11%. LOD: 0.74 ␮g kg−1
GC–ECD (300 ◦ C). Injector: with temperature gradient. Column: HP1 [13]
(25 m × 0.32 mm i.d.; 0.52 ␮m). Recovery: 88–93%. R.S.D. < 8%.
LOD: 0.001 mg kg−1
HPLC–DAD. Column: C18 (150 mm × 4.6 mm i.d., 5 ␮m). Mobile [5]
phase: acetonitrile–0.01 M TEA (pH 6.1 with 0.75 M H3 PO4 ) in
gradient mode at a flow-rate of 1 ml min−1 . Recovery: 63–100%.
R.S.D. < 8%. LOD: 0.2 mg kg−1
GC–MSD electronic impact (ionization potential 70 eV, ion source [33,40]
230 ◦ C, transfer line 250 ◦ C). Injector: splitless at 250 ◦ C. Column:
BP-1 (12 m × 0.22 mm i.d.; 0.25 ␮m). Recovery: 74–96. R.S.D. < 8%.
LOQ <0.04 mg kg−1 . Amitraz, bromopropylate, coumaphos and
fluvalinate. GC–ECD (250 ◦ C) and GC–FID (250 ◦ C). Injector:
splitless at 250 ◦ C. Column: RSL-200 (25 m × 0.25 mm i.d.; 0.25 ␮m).
Recovery: 75–93%. R.S.D. < 10%. LOQ <0.02 mg kg−1
LC–APcI–MSD in positive and negative modes (vaporized 350 ◦ C, [34]
drying gas 350 ◦ C, capillary voltage 4000 V, and corona current
discharged 4 ␮A in positive mode and 25 ␮A in negative mode).
Column: C18 (250 mm × 4.6 mm i.d.; 5 ␮m). Mobile phase:
methanol–water gradient at a flow-rate of 1 ml min−1 . Recovery:
14–102%. R.S.D. < 17%. LOD < 0.24 mg kg−1
HPLC–APcI–MSD in negative mode (vaporized 400 ◦ C, drying gas [32]
350 ◦ C, capillary voltage 4000 V, corona current discharged 25 ␮A).
Column: C18 (250 mm × 4.6 mm i.d., 5 ␮m). Mobile phase:
methanol–water gradient at a flow-rate of 0.8 ml min−1 . GC–MSD
electronic impact (70 eV ionization potential, 250 ◦ C Ta ion source,
200 ◦ C Ta transfer line, 230 ◦ C Ta analyzer). Injector: splitless at
220 ◦ C. Column: DB-5 (30 m × 0.25 mm i.d., 0.25 ␮m). Recovery:
73–98%. R.S.D. < 19%. LOQ <0.1 mg kg−1
GC–MSD electronic impact (70 eV ionization potential, 230 ◦ C Ta ion [38]
source, 150 ◦ C Ta analyzer). Injector: splitless at 280 ◦ C. Column:
ZB-5MS (30 m × 0.25 mm i.d.; 0.25 ␮m). Recovery: 86–101%.
R.S.D. < 10%. LOD < 0.006 mg kg−1
 R. Rial-Otero et al. / Talanta 71 (2007) 503–514
Table 2 (Continued )
Analytes/extraction procedure
Bromopropylate, coumaphos, fluvalinate. SPE. 10 g of honey diluted with
15 ml of ethanol–water (2:3, v/v) were passed through a C18
packed-column and the pesticides were eluted with 2 ml of ethyl acetate
and 2 ml of hexane
39 pesticides. SPE. 1 g of honey diluted with 2 ml of methanol were passed
through a florisil packed-column and the pesticides were eluted with
30 ml of n-hexane–dichloromethane (1:1, v/v)
Cymiazole. SPE. 20 g of honey diluted with 20 ml of 1% acetic acid in
water and passed through a SCX–SPE cartridge. The cartridge was
washed with 4 × 1 ml of methanol. Then, cymiazole was deprotoned by
passing 1 ml of phosphate buffer (0.01 M, pH 7.7) and the pesticide was
eluted with 1 ml of methanol
26 pesticides (OCPs and OPPs). SPE. 12 g of honey were diluted with
40–50 ml of methanol–water (70:30, v/v). A 5 ml aliquot of the solution
was passed through an ENV+ packed-column and the column was
washed with 2 ml of methanol–water (70:30, v/v). The pesticides were
eluted with 3 ml of ethyl acetate
16 pesticides. MSPD. 1–1.5 g of honey diluted with 1.5 ml of methanol
were transferred to a column filled with 2.5 g florisil and 1 g anhydrous
sodium sulphate. 5 ml of hexane–ethyl acetate (90:10, v/v) were added,
the column was closed and sonicated in ultrasonic water bath for 15 min.
The extractant was removed in a vacuum manifold and the extraction
was repeated
21 pesticides. SPME. The honey sample diluted with water (1:5) was
extracted with a PDMS fiber (100 ␮m) during 60 min at 70 ◦ C.
Desorption of pesticides at 260 ◦ C for 4 min
Bromopropylate, chlorfenvinphos, coumaphos, diazinon, ethion,
pirimiphos methyl, terbuphos. SPME. 0.5 g of honey diluted in 10 ml of
water was extracted with a PDMS fiber (100 ␮m) during 60 min under
stirring. Desorption of pesticides at 270 ◦ C for 3 min
Amitraz, bromopropylate, 2,4-dimethylaniline, coumaphos, cymiazole and
fluvalinate. SPME. 1 g of honey diluted in 9 ml of water and 50 ␮l of
ammonia 30% solution was extracted with a PDMS fiber (100 ␮m)
during 50 min under stirring. Desorption of pesticides at 250 ◦ C for 3 min
6 OPPs. SPME. 2.5 g of honey diluted in water (1:10) were extracted with
a PDMS fiber (100 ␮m) during 120 min under stirring at 900 rpm.
Desorption of pesticides in 1 ml of methanol for 15 min
11 OPPs. SPME. 23 ml of honey diluted in water (1:5) saturated with NaCl
were extracted with a sol–gel CROWN ETHER fiber (40 ␮m) during
60 min at a 55 ◦ C. Desorption of pesticides at 260 ◦ C for 4 min
6 OPPs. SBSE. 2.5 g of honey diluted with water (1:10) were extracted
with a PDMS Twister (10 mm × 1 mm) during 120 min under stirring at
900 rpm. Desorption of pesticides in 1 ml of methanol agitating for
15 min
509
Technique/comments/quality parameters Ref.
GC–ECD. Column: DB-5 (30 m × 0.25 mm i.d.; 0.25 ␮m). [39]
LOD < 0.003 mg kg−1
Acrinathrine, 3-phenoxybenzaldehyde. GC–FID (300 ◦ C). Injector: in [16,36]
splitless mode at 225 ◦ C. Column: a fused silica capillary column with
50% of phenyl methylpolysiloxane (30 m × 0.25 mm i.d.; 0.25 ␮m).
Recovery: 100%. R.S.D. < 3.6%. LOD < 0.01 mg kg−1 . 37 pesticides
GC–ECD (300 ◦ C) and GC–NPD (300 ◦ C). Injector: in splitless mode
at 200 ◦ C. Column: a fused silica capillary column with 50% of phenyl
methylpolysiloxane (60 m × 0.25 mm i.d.; 0.25 ␮m). Recovery:
97–102%. R.S.D. < 7%. LOD < 0.075 mg kg−1
HPLC–UV (265 nm). Column: ODS-2 (100 mm × 2.1 mm i.d., 3 ␮m). [30]
Mobile phase: acetonitrile–phosphate buffer (0.01 M, pH 2.5) with a
1% tetramethylammonium nitrate (80:20, v/v) at a flow-rate of
0.2 ␮l min−1 . Recovery: 89–94%. R.S.D. < 6%. LOD < 0.002 mg kg−1 .
HPLC–EC (WE potential 1.1 V). Column: ODS-2 (100 mm × 2.1 mm
i.d., 3 ␮m). Mobile phase: acetonitrile–phosphate buffer (0.01 M, pH
2.5) with a 1% tetramethylammonium nitrate (50:50, v/v) at a flow-rate
of 0.2 ␮l min−1 . Recovery: 55–60%. R.S.D. < 11%. LOD < 0.7 mg kg−1
22 OCPs and OPPs. GC–ECD (300 ◦ C). Injector: splitless at 210 ◦ C. [20,29]
Columns: 007–2 (50 m × 0.25 mm i.d.; 0.25 ␮m). GC–NPD. (250 ◦ C).
Injector: splitless at 200 ◦ C. Columns: HP-1 (12 m × 0.20 mm i.d.;
0.3 ␮m). Recovery: 63–98%. R.S.D. < 28%. LOQ <0.025 mg kg−1
(Ref. [29]). 26 pesticides. GC–ECD (300 ◦ C), GC–TSD (300 ◦ C) or
GC–PFPD (275 ◦ C). Columns: CP-Sil 8CB (25 m × 0.32 mm i.d.;
0.25 ␮m), CP-Sil 19CB (25 m × 0.32 mm i.d.; 0.20 ␮m). Recovery:
85–127%. R.S.D. < 27%. (Ref. [20])
16 pesticides. GC–ECD (270 ◦ C). Injector: 250 ◦ C. Column: HP-1 [42,43]
(30 m × 0.25 mm i.d.; 0.25 ␮m). Recovery: 80–113%. R.S.D. < 10%.
LOD < 0.015 mg kg−1 . (Ref. [42]). Pirimicarb, amitraz and 12 OPPs.
GC–NPD (270 ◦ C). Injector: 250 ◦ C. Column: HP-1 (30 m × 0.25 mm
i.d.; 0.25 ␮m). Confirmation by GC–MSD electronic impact
(ionization potential 70 eV; ion source 230; analyzer 150 ◦ C), equipped
with a HP-5MS column (30 m × 0.25 mm i.d.; 0.25 ␮m). Recovery:
60–112%. R.S.D. < 10%. LOD < 0.012 mg kg−1 (Ref. [43])
GC–ECD (300 ◦ C). Column: a fused silica capillary column with 50% [48]
of phenyl methylpolysiloxane (60 m × 0.25 mm i.d.; 0.25 ␮m).
Recovery: 81–115%. R.S.D. < 20%. LOD < 0.03 mg kg−1
GC–MSD electronic impact (transfer line 280 ◦ C). Injector: splitless at [51]
270 ◦ C. Columns: Rtx-5MS (30 m × 0.25 mm i.d.; 0.25 ␮m).
R.S.D. < 26%. LOD < 0.003 mg kg−1
GC–MSD electronic impact (transfer line 280 ◦ C). Injector: splitless at [51]
250 ◦ C. Column: PTE-5 (4 m × 0.25 mm i.d.; 0.25 ␮m). R.S.D. < 51%.
LOD < 0.01 mg kg−1
LC–APcI–MSD in positive and negative modes (vaporized 450 ◦ C; [49]
drying gas 350 ◦ C; capillary voltage 3500 V; corona current discharged
25 ␮A). Column: C18 (250 mm × 4.6 mm i.d., 5 ␮m). Mobile phase:
methanol–water gradient at a flow-rate of 0.7 ml min−1 . Relative
recovery: 3.6–7.6%. R.S.D. < 10%. LOD < 0.5 mg kg−1
GC–FPD (250 ◦ C). Injector: 270 ◦ C. Column: HP-5 (30 m × 0.32 mm [50]
i.d.; 0.25 ␮m). Relative recovery: 74–105%. R.S.D. < 15%.
LOD < 0.001 mg kg−1
LC–APcI–MSD: in positive and negative modes (vaporized 450 ◦ C; [49]
drying gas 350 ◦ C; capillary voltage 3500 V; corona current discharged
25 ␮A). Column: C18 (250 mm × 4.6 mm i.d.; 5 ␮m). Mobile phase:
methanol–water gradient at a flow-rate of 0.7 ml min−1 . Recovery:
40–64%. R.S.D. < 9%. LOQ <0.1 mg kg−1
510 R. Rial-Otero et al. / Talanta 71 (2007) 503–514
cases, the use of an organic modifier or sample lyophilization
should be considered. In addition, high cost of the technology
used to perform the extractions is other drawback.
3.3. SPE
In SPE the sample is passed through a cartridge or a packed-
column filled with a solid sorbent where the pesticides are
absorbed and then eluted with an organic solvent. This procedure
present several advantages: (i) it is less time consuming than SE
procedure, (ii) it decreases the use of toxic solvents, (iii) the
extraction efficiency is not hindered by the formation of emul-
sions, and (iv) offers the possibility of automation [29–31]. How-
ever, the main drawbacks of SPE procedure are: (1) high blank
values, (2) considerable performance variation between the
products offered by different manufacturers (3) lot to lot varia-
tion (4) treatment of large sample volumes is not possible in some
cases, and (5) SPE cartridges are normally constructed with plas-
tics, which can adsorb the analyte and also to increase the inter-
ferences in the analysis [31]. Before SPE takes place, the sample
must be diluted with water [14,17,32–34], methanol [16,35,36],
methanol–water [20,29,37,38] or ethanol–water [13,39] to facil-
ity the pass of the sample through the solid phase. The following
sorbents has been successfully used in SPE for pesticide extrac-
tion from honeys: (a) the reversed-phase C18 cartridge is by far
the most common choice done by researchers for the extrac-
tion of insecticides, acaricides, fungicides, herbicides, OCPs
and OPPs from honey [5,14,17,32–35,37–40]; (b) florisil sor-
bent has been used for pyretroids, OCPs and OPPs [16,36],
(c) ENV-cartridges for OCPs and OPPs pesticides [20,29], (d)
SCX-cartridges for the extraction of cymiazole [30] and (e) C8
cartridges for tau-fluvalinate [13]. The choice of one sorbent
or another depends on the analyte polarity and on the possible
co-extracted interferences. Sample pH can be critical to obtain
high yields of pesticide retention in the sorbent. Thus in some
cases, sample pH modification can be necessary in order to sta-
bilize the pesticides and increase their absorption in the solid
phase [5,30,35]. For instance, at very acid pH values the resid-
ual silanols of C18 cartridges are uncharged and interactions with
protonated and charged analytes, such as DPMF and cymiazole,
are weakened. In contrast, at basic pH values the amine groups of
the analytes are uncharged and ionic interactions with the unpro-
tonated silanol are also more difficult as in the case of fluvalinate.
As additional examples, amitraz recovery improved when pH
increase from 2 to 10 probably due to its rapid degradation at low
pH. On the contrary, coumaphos is unstable in basic media and its
recovery decreases when pH increases [5]. Once the pesticides
have been retained in the SPE cartridges, are then eluted with
an organic solvent such as acetone [17], dichloromethane [13],
ethyl acetate [29], hexane [35,37], methanol [14,30], tetrahy-
drofurane [5] or mixtures of hexane–ethyl acetate [38,39],
hexane–dichloromethane [16,33,36,40], and methanol–water
[20] or methanol–ethyl acetate–dichloromethane [32,34]. Pes-
ticide polarity and the sorbent used for SPE extractions are
directly related to the choice of the best elution sorbent. SPE
and SE were compared for pesticide extraction. Although sim-
ilar recoveries and detection limits were obtained, SPE gave
better precision (<4%) and less non-desired co-extracted com-
pounds, which was reflected in better chromatograms [16,17].
However, the SPE approach requires a careful knowledge of the
system in order to choose the best sorbent for the application
to be developed. Thus, Bernal et al. [16] compared SE with a
mixture of benzene–isopropanol, with SPE on ODS cartridges
or florisil packed-columns for the isolation of acrinathrine and
its main metabolite, 3-phenoxybenzaldehyde, from honey. They
observed that ODS cartridges always produce recoveries lower
than 58% due to the low solubility of pyretroids in water.
3.4. MSPD
MSPD is an extraction and clean-up technique that was first
developed by Barker and co-workers in 1989 to avoid the gen-
eral drawbacks of SE and SPE for the extraction of analytes
from solid or semi-solid samples [41]. This technique required
less time and solvent than SE and provides similar results. In
addition, it avoids the previous diluting steps for solid or semi-
solid samples in order to extract them by SPE. In the MSPD the
sample is mixed with an adsorbent, generally florisil or C18 , and
then transferred to a column. The sorbent has several functions:
(1) works as abrasive compound breaking the physical struc-
ture of the sample, (2) adsorbs the compounds of the matrix,
(3) it works as a solid support for filling the column and (4)
allows the fractionation of the sample. The column is finally
eluted with an appropriate solvent and then the extract can be
analyzed directly. Interferences, such as pigments or other polar
compounds, are retained on the sorbent. This technique was
successfully assessed for the extraction of pyretroids, OCPs,
OPPs, and acaricides from honey [42,43] in the following man-
ner: the sample was diluted in methanol and transferred to a
column filled with florisil and anhydrous sodium sulphate. Then
the pesticides were eluted with ethyl acetate. This method pro-
vides recoveries between 60% and 113% and detection limits
lower than 0.015 mg kg−1 .
3.5. SPME
SPME was first developed by Pawliszyn and co-workers
[44–47], and has become popular for the analysis of organic
compounds because it combines sampling and pre-concentration
in a single step. In this technique a fused silica fiber coated
with a polymeric film is immersed into the sample. The pesti-
cides are adsorbed into the stationary phase and later thermically
desorbed into the injection port of a gas chromatograph (see
Fig. 2). SPME eliminates the problems associated with SPE,
as described previously, while retaining the following advan-
tages: (i) solvents are completely eliminated, (ii) blanks are
greatly reduced, (iii) extraction time is reduced to a few min-
utes, (iv) it provides good results over a wide range of analyte
concentrations, (v) it does not require complete removal of the
analyte from the liquid matrix, and (vi) can be easily automated
[4,31]. SPME was evaluated for the extraction of acaricides,
OCPs, OPPs and ONPs from honey [48–51]. Obviously, a con-
cern in this methodology is the composition of the fibers. Thus,
most pesticides have been extracted with polydimethylsilox-
 R. Rial-Otero et al. / Talanta 71 (2007) 503–514 511

Table 3
Levels detected for pesticide residues in commercial honey
Year Origin No. samples Pesticides Levels Ref.
2005 Spain 111 OCPs 0.2–8 ␮g kg−1 [29]
OPPs 5–12 ␮g kg−1

2004 Spain 11 Dichlofluanid 6–11 mg kg−1 [38]

Athalfluralin and Triallate <4.4 mg kg−1 [38]
15 Chlorpyriphos-methyl 2 mg kg−1 [49]
2003 Spain and 50 OCPs 0.01–4.31 mg kg−1 [32]
Portugal OPPs 0.01–0.23 mg kg−1
Carbamates 0.003–0.64 mg kg−1
2002 France 320 Fluvalinate 0.01–0.026 mg kg−1 [11]
Coumaphos 0.11–0.26 mg kg−1
2001 Italy 60 Fluvalinate <0.09 ␮g kg−1 [22]
1997 Spain 101 Bromopropylate 0.005–0.06 mg kg−1 [40]
Fluvalinate 0.01–0.04 mg kg−1
Jordan 26 OCPs 0.001–0.37 mg kg−1 [21]
OPPs <0.2 mg kg−1
Fluvalinate <0.13 mg kg−1

ane (PDMS, 100 ␮m) fiber [48,49,51]. However, other kinds
of fibers have been successfully tried. For instance, the use
of a sol–gel CROWN ETHER® fiber (40 ␮m) was proposed
for remove 11 OPPs from honey with good relative recoveries
(74–105%) and low detection limits (<0.001 mg kg−1 ) [50]. The
analytical performance obtained from various materials can be
remarkably different, as it was demonstrated by Jimenez et al.
[48], who compared two different fibers, namely polyacrylate
(85 ␮m), and PDMS (7 and 100 ␮m). The PDMS showed the
following advantages: (i) enhanced reproducibility, (ii) lower
detection limits, (iii) extended linearity, (iv) improved correla-
tion coefficients, (v) low extraction time, and (vi) better chro-
matograms. As for the other aforementioned procedures, sample
pH is of great concern. Thus, for the determination of cymi-
azole by SPME the addition of 50 ␮l concentrated ammonia
(30%) was necessary to increase the pH of the sample, displac-
ing in this way the protonation equilibrium toward the formation
of non-protonated species of cymiazole, that are then readily
absorbed by the PDMS fiber [51]. In other cases, the pH vari-
ation can led to worst results, such as the ones reported by
Volante et al., and dealing with the simultaneous determina-
tion of coumaphos and cymiazole. In this case, the addition of
ammonia to increase the recuperation of cymiazole produces a
reduction in the intensity of the coumaphos peak that may be
explained by the hydrolysis reaction with reversible opening of
the pyrone ring which decreases its adsorption to the SPME fiber
[51].
3.6. SBSE
SBSE is relatively new technique, theoretically similar to
SPME. It has been used with success for the extraction of organic
compounds from aqueous food, biological and environmental
samples. In SBSE the sample is stirred for a given time with a
stir bar coated with a sorbent, until the analyte reach the equi-
librium between the polymer and the aqueous phase according
to their distribution constant. Then the analytes are desorbed by
high temperatures into the injector port of the GC or by liquid
removal for HPLC analysis. The most important advantages of
SBSE are the same than for SPME, however, higher recoveries
are obtained with SBSE [25]. To date, the only sorbent used
for SBSE is PDMS, although the development of new stir bars
coated with polar sorbents is predicted in literature. This limita-
tion constitutes SBSE Achilles Heel. Applicability of SBSE to
determine pesticides in fruit and vegetables has been success-
fully proved [52–54]. However, by now, only Blasco et al. have
applied this technique for the extraction of six OPPs from honey
[49]. These authors also compared the use of SBSE with SPME
and concluded that although linearity and precision obtained by
both techniques are similar, SBSE is more accurate, sensitive
and the effect of honey matrix in the quantification is lower than
SPME.
4. Chromatographic separation
Chromatographic-based techniques are used to identify and
quantify pesticides residues in honey. As can be seen in
Table 2, gas chromatography (GC) has been widely used for
pesticide determination/quantification with the following detec-
tors: (1) electron capture detector (ECD) [2,13,15,17,19–21,
29,35,36,39,40,42,43,48]; (2) nitrogen–phosphorus detector
(NPD) [18,19,21,29,36,43]; (3) flame ionization detector (FID)
[16,40]; (4) atomic emission detector (AED) [12,18]; (5) mass
spectrometry detection (MSD) [2,12,17,22,32,33,38,43,51]; (6)
flame photometric detector (FPD) [50]; (7) thermoionic-specific
detector (TSD); or (8) pulsed flame photometric detection
(PFPD) [20]. Most of them are specific detectors, for exam-
ple ECD is used for compounds with electronegative atoms
whilst NPD is used for compounds with nitrogen or phosphor
in their structures. However, others are non-specific detectors,
for instance FID. MSD is the universal and non-specific detector
that allows not only to detect and to quantify the analytes present
512 R. Rial-Otero et al. / Talanta 71 (2007) 503–514
in the sample, but also to identify these compounds on basis on
their structure.
The choice of the column is very important in pesti-
cide analysis. In this way, the stationary phase should be
selected as a function of the pesticide polarity. Non-polar
or medium-polar columns (100% polydimethylsiloxane–50%
phenylmethylpolysiloxane) are the most used columns for pes-
ticide analysis in honey as can be seen in Table 2. The use
of 14% cyanopropyl diphenyldimethylpolysiloxane column was
also proposed for multiresidue analysis [20], whilst other authors
preferred to use a special column designed for OCPs analysis
[2]. Moreover, other column parameters such as length, inner
diameter or film thickness can be optimized as a function of the
number of pesticides that must be determined simultaneously.
For some pesticides determination by GC is impossible due
to their low thermal stability and/or their insufficient volatil-
ity without further chemical derivatization. For such cases the
alternative is liquid chromatography. As an example, fluvali-
nate residues can be determined by gas chromatography with
ECD or NPD detectors [15,19,21,22,39,40] but fluvalinate is
decomposed readily in the GC injector and/or column due to
the high temperatures. As consequence, fluvalinate determina-
tion is developed by HPLC–UV [26]. The most widely used
HPLC detectors for pesticide analysis are diode array (DAD)
[5,11,37] and UV [14,26,30]. Due to its versatility, high selectiv-
ity, and spectral evidence of individual solutes, MSD detection
employing atmospheric pressure ionization (API), in positive
and negative modes, is becoming the detection system of choice
for most researchers [32,34,49]. However, the high price of MSD
along with their expensive maintenance considerably boots the
expansion of the aforementioned detection system. The use
of others detectors such as fluorescence detector (FLD) [23]
and amperometric detector (EC) [30] was only reported for the
analysis of benomyl, carbendazim and cymiazole. In HPLC, pes-
ticides separation is generally carried out using reversed-phase
chromatography in C18 column (4.6 mm i.d.). Nevertheless the
column type is always critical. For instance, chromatograms of
cymiazole from ODS columns give peaks with tailing because
of the interaction of cymiazole with residual silanol groups on
the silica support [30]. For solve this problem, phosphate buffer
with a 1% of tetramethylammonium nitrate or good en-capped
ODS columns can be used. It is true that in the presence of com-
pounds, such as tetramethylammonium salts, the influence of
silanol groups is negligible but for strongly basic compounds
this effect is not completely removed [30]. The development
of liquid chromatography with narrow-bore (2.1 mm i.d.) and
microbore columns (0.32 mm i.d.) has led (i) to increase the
sensitivity and efficiency of the liquid chromatographic meth-
ods, (ii) to decrease of amount of solvents used, (iii) to reduce the
amount of sample required, and (iv) to produce an easy coupling
with other techniques [37]. As an example, Gomis et al. proposed
the use of small-bore ODS columns for the determination of
bromopropylate, coumaphos, 4,4 -dibromobenzophenone, flu-
valinate and cymiazole in order to decrease the detection limits
at levels comparable with those obtained by GC–ECD [30,37].
The mobile phase used in HPLC consists frequently in mixtures
of acetonitrile–water or methanol–water but, in some cases, the
analyte properties can lead to modifications of the mobile phase
pH. For instance, the very instability of amitraz in acid medium
makes necessary the use of the aqueous mobile phase at pH 9
[11]. In the determination of nine acaricides in honey, Korta
et al. pointed out that the mobile phase pH has to be higher
than 6 to improve the resolution between the most polar ana-
lytes. Nevertheless, high pH values produced more drift in the
baseline, and the authors selected a mobile phase with pH 6.1
[5].
Some final considerations about matrix effects. As it was
stated above, the honey matrix can affect the extraction effi-
ciency for pesticides [20,49,50]. In addition, matrix can also
affect the detector by increasing or decreasing its response. Thus,
for the same concentration, different intensity can be obtained
from standards and treated samples, even with intensity cleaning
procedures [29,36,38,39,51]. To avoid these problems, recovery
trials and quantitative analysis of different pesticides in honey
must be carried out using fortified blank samples.
5. Pesticide levels
Levels of pesticides cited in literature in commercial honeys
are summarized in Table 3. The high presence of OCPs (mainly
␥-HCH, ␣-HCH, ␤-HCH, DDT, DDD, DDE, and isomers) in
honey must be emphasized, because OCPs have been restricted
or banned for agriculture use since 1978 in the USA and Europe,
due to their persistence and bioaccumulation in the environ-
ment. However, these pesticides are still frequently found in
soils, from which they continue to cycle through the environ-
ment [32]. In any case, decreasing content is the tendency in
OCPs levels: from 370 to 8 ␮g kg−1 from 1997 to 2005, respec-
tively. Acaricides used to combat varroatosis or ascospheriosis,
bromopropylate, coumaphos and fluvalinate were found at lev-
els of 5–60, 110–260, and <1–130 ␮g kg−1 , respectively. OPPs
were found in honey samples from Jordan, Spain and Portugal
at levels between 3 and 64 ␮g kg−1 . Other pesticides such as
athalfluralin, chlorpyriphos-methyl, dichlofluanid, triallate and
some carbamates were also found. Special mention should be
made to the four ecological honeys analyzed in Spain in 2003
[32]. Although named “ecological” methidathion, methiocarb
and OCPs were found in concentrations ranging from 0.03 to
1.83 mg kg−1 .
6. Future trends
In the next future more research is expected in the sam-
ple treatment of pesticides in honey in two key issues. On
the one hand SBSE will be further investigated as the ultimate
methodology for pesticide extraction due to its inherent prop-
erties previously commented in Section 3.6. On the other hand,
versatile analytical techniques, such as MALDI–TOF–MS are
nowadays becoming available to more analytical laboratories.
Little has been done yet to improve this instrumentation for pes-
ticide identification, although some promising approaches have
been recently published [55].
 R. Rial-Otero et al. / Talanta 71 (2007) 503–514
7. Conclusions
Pesticide standards must be protected from light and the sol-
vent used to prepare them must be carefully chosen based on
literature or experimentally checked in order to avoid standards
degradation.
pH is an important parameter in the detection of pesticides
in honey since some of them are degraded at low or high pH.
Because of this the pH value during the extraction must be care-
fully selected.
Solvent extraction and SPE are the techniques most com-
monly used for the extraction of pesticides from honey. Nev-
ertheless, in the last years new alternative techniques such
as SPME and SBSE are used with good results. These new
approaches require less organic solvents, are easy of implemen-
tation, and allow high sample throughputs.
Identification and quantification of pesticide residues in
honey is generally carried out by gas chromatography. How-
ever, for either thermal unstable compounds or compounds with
low volatility liquid chromatography must be chosen.
Calibration curves with fortified blanks must be used to avoid
matrix effects in the quantification of pesticides.
Trazability of some pesticides in honey must be conducted in
samples directly taken from beehives, since pesticides such as
amitraz or chlordimeform degrade quickly, leading to degrada-
tion products. For some pesticides, these degradation products
must to be identified rather than the original compound, when
the sample has been stored for a long time.
Different pesticides residues were found in honey samples
produced in France, Jordan, Italy, Portugal, Spain and Switzer-
land. OCPs, OPPs and the acaricides fluvalinate, coumaphos
and bromopropylate are the most common pesticides detected.
Two OPPs, methidation and methiocarb, were also detected in
ecological honey from Spain.
Acknowledgement
Raquel Rial acknowledges the post-doctoral grant given by
the Direccion Xeral de Investigación, Desenvolvimento e Inno-
vación from Xunta de Galicia from Spain.
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