Ciência e Agrotecnologia, 42(1):51-57, Jan/Feb.

2018
http://dx.doi.org/10.1590/1413-70542018421021117

LC-MS/MS analysis of neonicotinoid insecticides: Residue

findings in chilean honeys
LC-MS/MS análises de inseticidas neonicotinoides: Resíduos
encontrados em méis chilenos

Raquel Bridi1*, Arturo Larena1, Paula Núñez Pizarro1, Ady Giordano1, Gloria Montenegro2

1
Pontifícia Universidad Católica de Chile, Facultad de Química, Santiago, Chile
2
Pontifícia Universidad Católica de Chile, Facultad de Agronomía e Ingeniería Forestal, Santiago, Chile
*
Corresponding author: [email protected]
Received in August 2, 2017 and approved in October 17, 2017

ABSTRACT
Neonicotinoids are a relatively new generation of insecticides that have been used for control of pests such as aphids, leafhoppers and
whiteflies. This paper presents for the first time a determination of residues of four neonicotinoid insecticides (acetamiprid, thiamethoxam,
thiacloprid and imidacloprid) in Chilean honey using QuEChERS extraction and UHPLC-MS/MS analysis. The limits of detection and
quantification found for all analytes ranging from 0.34 to 1.43 μg kg-1 and from 0.30 to 4.76 μg kg-1, respectively. The extraction using
QuEChERS method provided recoveries over 79% and the precision showed coefficient of variation lower than 20%. These data are in
agreement with the international criteria that recommend general recovery limits of 70 - 120%. Of the 16 samples analyzed, in three
honey samples neonicotinoids pesticides were detected. These three samples were collected from the same geographical area (Rengo).
Fruit and grain production characterize the province of Rengo. The analysis of the botanical origin of these honeys showed the absence
of pollen grains of crops and the majority presence of pollen grains of weeds such as Medicago sativa, Galega officinalis and Brassica rapa,
which could be associated with crops. Although the residue levels found were low, the results also confirm the actual occurrence of a
transfer of neonicotinoid insecticides from exposed honeybees into honey.

Index terms: QuEChERS; pesticide residues; food analysis.

RESUMO
Os neonicotinóides são uma geração relativamente nova de inseticidas que tem sido utilizado para o controle de pragas, como pulgões,
cigarrinhas e moscas brancas. Este artigo apresenta pela primeira vez a determinação de resíduos de quatro inseticidas neonicotinóides
(acetamiprida, tiametoxam, tiaclopride e imidaclopride) em méis chilenos empregando para extração o método QuEChERS seguido de
UHPLC-MS/MS. Foram encontrados limites de detecção e quantificação aceitáveis para todos os analitos variando de 0,34 a 1,43 μg kg-1
e de 0,30 a 4,76 μg kg-1, respectivamente. A extração usando o método QuEChERS proporcionou uma recuperação maior que 79% e a
precisão do método não excedeu um coeficiente de variação de 20%. Esses dados estão de acordo com os critérios internacionais que
recomendam limites gerais de recuperação de 70 - 120%. Das 16 amostras de mel analisadas foram detectados pesticidas neonicotinoides
em três delas. Essas três amostras foram coletadas na mesma área geográfica (Rengo), a qual se caracteriza pela produção de grãos e
frutas. A análise da origem botânica desses méis mostrou a ausência de grãos de pólen de espécies cultivadas nesta zona, porém mostrou
uma presença majoritária de grãos de pólen de ervas daninhas, como Medicago sativa, Galega officinalis e Brassica rapa, as quais podem
estar associadas a estes cultivos. Embora os níveis de resíduos neonicotinoides encontrados neste estudo são baixos, os resultados
confirmam a ocorrência atual de uma transferência destes inseticidas ao mel das de abelhas expostas.

Termos para indexação: QuEChERS; resíduos de pesticidas; análise de alimentos.

INTRODUCTION CropScience), clothianidin (Bayer CropScience and

Sumitomo), thiamethoxam (Syngenta), acetamiprid
Neonicotinoid pesticides have become the most (Nippon Soda), nitenpyram (Sumitomo), and dinotefuran
widely used class of insecticides worldwide, with large- (Mitsui Chemicals) (Simon-Delso et al., 2015). Their use
scale applications ranging from plant protection (crops, has increased considerably since the early 1990s and they
vegetables, fruits), veterinary products and biocides represent one of the fastest growing types of insecticides
to invertebrate pest control in fish farming. Seven put on the market since the launch of pyrethroids (Tanner;
neonicotinoid compounds are available commercially Czerwenka, 2011; Yáñez et al., 2013). The wide application
worldwide: imidacloprid and thiacloprid (Bayer of these insecticides is attributed to their selective mode of

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52 BRIDI, R. et al.

action at low doses, the ease and flexibility with which they Supelco (Bellefonte, PA, USA). Acetonitrile and methanol
can be applied, their long persistence, and their systemic were HPLC grade and supplied by Merck (Darmstadt,
nature (Aliouane et al., 2009; Blacquière et al., 2012; Germany). Water was purified in a Milli-Q system
Bonmatin et al., 2015). However, various adverse effects (Synergy, Millipore®, Darmstadt, Germany).
on the environment have been reported, which occur via a
Standards
number of routes including dust generated during riling of
dressed seeds, contamination and accumulation in arable Individual standard stock solutions of 5 mg mL-1
soils and soil water, run off into waterways, and uptake for each analyte were prepared in acetonitrile and stored
of pesticides by non-target plants through their roots or at -80 °C. The stock solutions were diluted and mixed
dust deposition on leaves. This provides multiple paths with acetonitrile to obtain a mixture working solution of
for chronic (and acute in some cases) exposure of non- all investigated analytes of 0.1 mg L-1. A calibration curve
target animals. Neonicotinoids act in a very specific way was prepared by dilution in acetonitrile at concentrations
as agonists on the postsynaptic nicotinic acetylcholine between 0.5 to 45 µg L-1.
receptor of the insect’s central nervous system, causing a
Honey collection
blockage of signal transmission (Yáñez et al., 2013). Since
the neonicotinoids block a specific neural pathway that is Sixteen honey samples proceeded from apiaries
more abundant in insects than in warm-blooded animals, located in Malloa, Placilla, San Fernando, San Vicente,
these insecticides are selectively more toxic to insects Rengo, Peralillo and Palmilla in the VI Region (Libertador
than mammals (Decourtye; Devillers, 2010; Jovanov et Bernardo O’Higgins Region) of Chile were collected
al., 2015). In humans, the effects of chronic neonicotinoid between 2013 and 2015. The botanical origin of the
pesticide exposure on health are still little known (Cimino honeys was determined according to Chilean regulation
et al., 2017). Pollinators are exposed through direct (Montenegro et al., 2008). Ten grams of honey were
contact with dust during drilling, consumption of pollen, diluted in 10 mL of distilled water, and centrifuged at
nectar, or guttation drops from seed-treated crops and 2,500 rpm for 5 minutes. The supernatant was eliminated
water (Bonmatin et al., 2015). Different studies have and the sedimented pollen re-suspended in distilled water
demonstrated that sub lethal amounts of neonicotinoids (0.1 mL). Five preparations from each honey sample were
alone or combined with other pesticides, such as fungicides analyzed using optical microscopy and the pollen grains
(Iwasa et al., 2004) may cause disorientation, reduced from each sample were identified using the palinoteque
communication, impaired learning and memory, reduced and reference bibliography.
longevity and disruption of honeybee brood cycles Matrix fortification
(Farooqui, 2013; Pisa et al., 2015). Furthermore, residues
of these insecticides may be found in bee products such The matrices used in this study were uncontaminated
as honey, pollen, beeswax, and propolis (Jovanov et al., honeys collected from beehives unexposed to pesticide
2015; Kasiotis et al., 2014; Tanner; Czerwenka, 2011). within an adequate perimeter. Five grams of each
For different neonicotinoids the maximum residue limit in matrix was fortified with standards of the four studied
honey has been set by the European Union (EU) to range neonicotinoids at 5, 10, 20, 50, and 100 μg kg-1 by adding
the appropriate amount of the 10 mg L-1 analyte mixture
from 10 to 200 μg kg-1. In this view, this paper presents
working solution.
for the first time a determination of residues of four
neonicotinoid insecticides (acetamiprid, thiamethoxam, QuEChERS type method
thiacloprid and imidacloprid) in Chilean honey using
QuEChERS extraction and UHPLC-MS/MS analysis. Five grams of honey (blank or spiked with
standard analyte solutions), 10 mL of water, and 10 mL of
acetonitrile were mixed in a 50 mL centrifuge tube, which
MATERIAL AND METHODS
was then vigorously shaken by hand until a homogeneous
solution was obtained. A mixture of SupelTMQue Citrate
Chemicals and reagents
(Supelco) containing magnesium sulfate, sodium chloride,
Acetamiprid, thiamethoxam and thiacloprid sodium citrate tribasic dihydrate, and sodium citrate
were purchased from Sigma-Aldrich Laborchemikalien dibasic sesquihydrate was added to the tube. The tube
GmbH (Steinheim, Germany). Imidacloprid, SupelTMQue was shaken vigorously by hand for 1 min and centrifuged
Citrate and SupelTMQue PSA/C18 were purchased from for 5 min at 3000 g and 10 °C. An aliquot of 6 mL of

Ciência e Agrotecnologia, 42(1):51-57, Jan/Feb. 2018

LC-MS/MS analysis of neonicotinoid insecticides: residue findings in chilean honeys 53

the acetonitrile phase was transferred into a Pyrex tube where at least 45% or more pollen grains found in it
containing SupelTMQue PSA/C18 (Supelco) containing belong to the same species; bifloral honeys are those
magnesium sulfate, PSA and discovery C18. The tube was where pollens from two species are dominant within
vigorously shaken by hand for 1 min and centrifuged for 5 the total pollen grains, so that, as a whole, both species
min at 3000g and 10 °C. Two milliliters of the supernatant cover more than 50% of the total pollen grains, and
was evaporated to dryness using a stream of nitrogen. The there is not a difference higher than 5% among them
residue was redissolved in 1mL of methanol:water 20:80 and; polyfloral honeys are those where no species
(v/v) and subjected to LC-MS/MS analysis. reaches at least 45% of the total pollen grains, nor two
of them covers more than 50% of the said total. In the
UHPLC-MS/MS analysis analyzed honey we found nine monofloral, two bifloral
Quantification was performed in a Triple Quad™ and five polyfloral (Table 2). Samples from two
4500 System coupled with an Eksigent Ekspert Ultra native species were found Retanilla trinervia (tevo)
LC 100-XL system. Chromatographic separation was and Quillaja saponaria (quillay) while the species
achieved in a Inersil ODS-4 column (2.1 x 100 mm, 3 introduced mainly found were Galega officinalis
µm, GL Sciences) at 40 °C with a mobile phase of 0,1% L. (galega), Brassica rapa (yuyo), Medicago sativa
formic acid (A) and acentonitrile (B). Initial mobile (alfafa) and Melilotus indicus.
phase concentration was 10% B increased to 80% in According to previous data shown in the report
3 min, and kept constant for 8 min at a 0.4 mL/min of the Red de Acción en Plaguicidas y sus Alternativas
flow rate, with injection volume 10 μL. Electrospray de América latina (RAP-AL) the three most commonly
ionization was performed in positive mode. Fragmentor used insecticides in this area (O’Higgins Region -Chile),
voltage and collision energies were optimized for each are acetamiprid, thiamethoxam and imidacloprid
analyte during infusion of the pure standard, and the (CIAP, 2012). In this study, four neonicotinoid
most abundant fragment ion was chosen for the selected insecticides were investigated (acetamiprid, thiacloprid,
reaction monitoring. Quantitative analysis was carried out thiamethoxam and imidacloprid) in Chilean honey
using multiple reaction monitoring (MRM) mode, using samples. The gradient system (formic acid 0.1% and
a first transition for quantification and a second transition acentonitrile) was applied to separate the four pesticides
for identification purpose. For the proposed method, the as independent peaks. Retention times (t R ) were
most intense characteristic MRM transitions were chosen determined individually and selected ion monitoring
for each analyte (Table 1). (SIM) of neonicotinoid insecticides with UHPLC–MS
are presented in Table 1.
RESULTS AND DISCUSSION The linearity of the calibration curve of each
pesticide was established by plotting UHPLC response
A melissopalinological assay was used to area ratio versus concentration. The analytes showed
determine the botanical origin of the honeys samples linear behavior in the studied concentration range of 0.5
selected for this study. In Chile, according the official – 45 μg L-1. The correlation coefficient (𝑟2) was found
policy (NCh2981.Of2005) established by the Standards to be ≥0.994 for all pesticides. Limit of quantification
Division of the National Institute for Standardization (LOQ) and limit of detection (LOD) were calculated for
honey can be classified according to three types of each insecticide and are presented in Table 3. A sample
botanical origins: monofloral, bifloral, or polyfloral was considered positive when residue levels were above
(Montenegro et al., 2008). Monofloral honeys are those the LOQ.

Table 1: Retention times, molecular weight and monitored ions of neonicotinoid insecticides with LC–MS.

Insecticide tR Molecular mass Quantification ion (m/z) Identification ion (m/z) DPa (V) CEb (V)
Acetamiprid 4.89 222.7 126.0 89.9 86 86
Thiamethoxam 4.00 291.7 210.9 180.9 61 61
Thiacloprid 5.40 252.7 125.9 185.9 76 76
Imidacloprid 4.62 255.7 209.0 175.0 66 66
a
Declustering potential; Collision energy.
b

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54 BRIDI, R. et al.

Table 2: Floral composition of honey samples studied from VI Region, Chile.

Apiaries localization Types of botanical origins Clasification Predominant species % pollen grains
1 Malloa Monofloral Native Quillaja saponaria 74.9
2 Placilla Monofloral Non- Native Galega officinalis 59.9
3 San Fernando Monofloral Native Quillaja saponaria 46.6
4 San Vicente Monofloral Native Quillaja saponaria 46.5
5 San Vicente Monofloral Native Quillaja saponaria 46.2
6 San Vicente Polyfloral Non- Native Galega officinalis 24.3
7 San Vicente Polyfloral Native Retanilla trinervia 32.6
8 San Vicente Monofloral Native Quillaja saponaria 46.2
9 Rengo Bifloral Non- Native Galega/Medicago 37.0
10 Rengo Polifloral Native Luma apiculata 34.8
11 Rengo Bifloral Non- Native Galega/Medicago 37.8
12 Peralillo Monofloral Non- Native Galega officinalis 54.0
13 Palmilla Monofloral Non- Native Melilotus indicus 50.3
14 San Vicente Polyfloral Non- Native Melilotus indicus 43.4
15 San Vicente Polyfloral Non- Native Brassica rapa 28.4
16 San Vicente Monofloral Native Quillaja saponaria 91.8

Table 3: Limits of detection and quantification for four fortification level of 100 μg kg-1 (Table 4); with coefficient
neonicotinoid insecticides in honey samples. of variation lower than 20%. These data are in agreement
µg kg-1 honey
with the criteria of document no. SANCO/12495/2011,
Insecticide that recommend general recovery limits of 70-120%
LOD LOQ
(SANCO/12571/2013, 2013).
Acetamiprid 0.34 1.14
Thiamethoxam 0.11 0.30
Table 4: Recovery rates for four neonicotinoid insecticides.
Thiacloprid 1.43 4.76
Recovery (%)
Imidacloprid 0.47 1.56 Insecticide
20 µg/Kg 50 µg/Kg 100 µg/Kg
(LOQ) Limit of quantification; (LOD) limit of detection.
Acetamiprid 79 ± 1.4 87± 3.4 100 ± 2.4
Thiamethoxan 82 ± 3.6 87± 2.5 100 ± 2.5
The QuEChERS methodology is composed of an
Thiacloprid 81 ± 5.7 84 ± 3.3 101 ± 1.1
extraction step with acetonitrile and partitioning using
MgSO4, followed by dispersive solid phase extraction Imidacloprid 85 ± 0.3 81 ± 6.4 99 ± 4.0
using primary-secondary amine (PSA). Different
approaches have been used according anaytes and matrix.
In honey samples, for neconictonoid determination, higher The validated method was employed for the
recoveries were obtained when adding citrate salts to the analysis of sixteen honey samples obtained from
extraction methodology, that when using the original apiaries situated in different localities of VI Region,
QuEChERS procedure with anhydrous magnesium sulfate Chile and harvested in the summer months of the years
and sodium chloride (Paradis et al., 2014). The extraction 2013, 2014 and 2015. All honey samples were analyzed
using this method for honey samples provided high in triplicate. The main targets of the analyses were
recovery which ranged from 79% (acetamiprid) to 85% to examine the presence of neonicotinoid insecticide
(thiamethoxan) for the fortification level of 20 μg kg-1 and residues in Chilean honey. Figure 1 demonstrates the
from 99% (thiacloprid) to 101% (thiamethoxan) for the UHPLC-MS chromatogram of a honey sample containing

Ciência e Agrotecnologia, 42(1):51-57, Jan/Feb. 2018

LC-MS/MS analysis of neonicotinoid insecticides: residue findings in chilean honeys 55

neonicotinoid insecticides. The major peak at 5.40 min geographical area (Rengo, Chile). The province of
represents thiacloprid and the peak at 4.89 represents Rengo is characterized by a high incidence of grain
acetamiprid. crops such as cereals, legumes, raps, sunflowers and
Ta b l e 5 s h o w s t h e s u m m a r y r e s u l t s o f beets, and fruit crops such as almonds, plums, pears,
neonicotinoid residues detected in all samples that apples and grape. In addition to other provinces in the
were analyzed in this study. Of the 16 samples analyzed, VI Region, Rengo makes up an important percentage
in one sample residues of all pesticides were detected, of vineyard areas of the country. In the botanical
while in a second sample only acetamiprid, thiacloprid, origin of these honeys, the presence of pollen grains
and imidacloprid residues were found, and finally of these crops was not identified but was identified the
in a third sample only acetamiprid and thiacloprid majority presence of pollen grains of weeds that could
residues were detected. The three samples with detected be associated with these crops, such as Medicago sativa,
insecticide residues were collected from the same Galega officinalis and Brassica rapa (Table 2).

Figure 1: UHPLC-MS/MS chromatogram of a honey sample containing thiacloprid (peak at 5.40 min) and
acetamiprid (peak at 4.89 min).

Table 5: Concentrations of neonicotinoid measured in honey samples from the VI Region of Chile.

Concentration µg kg-1 honey

Sample
Acetamiprid Thiametoxan Thiacloprid Imidacloprid
1 n/d n/d n/d n/d
2 n/d n/d n/d n/d
3 n/d n/d n/d n/d
4 n/d n/d n/d n/d
5 n/d n/d n/d n/d
6 n/d n/d n/d n/d
7 n/d n/d n/d n/d
Continue...

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56 BRIDI, R. et al.

Table 5: Continuation...
Concentration µg kg-1 honey
Sample
Acetamiprid Thiametoxan Thiacloprid Imidacloprid
8 n/d n/d n/d n/d
9 78 < LOQ 63 7
10 34 n/d 31 n/d
11 14 n/d < LOQ < LOQ
12 n/d n/d n/d n/d
13 n/d n/d n/d n/d
14 n/d n/d n/d n/d
15 n/d n/d n/d n/d
16 n/d n/d n/d n/d
nd: non detected; < LOQ: residues of this pesticide above the LOD and below the limit of quantification.

CONCLUSIONS BLACQUIÈRE, T. et al. Neonicotinoids in bees: A review

on concentrations, side-effects and risk assessment.
In this work, an UHPLC-MS/MS analytical method Ecotoxicology, 21(4):973-992, 2012.
based on QuEChERS sample pretreatment procedures was
used for the determination of selected neonicotinoids in BONMATIN, J. M. et al. Environmental fate and exposure;
honey. Analysis of 16 honey samples from the VI Region neonicotinoids and fipronil. Environmental Science and
of Chile showed the presence of neonicotinoid residues Pollution Research, 22(1):35-67, 2015.
in three samples. These samples were collected from the CIAP. Disappearance of bees and pesticide residues
province of Rengo. This province is characterized by grain in honey. Situation in the region of O’Higgins.
and fruits crop, besides being an important vineyard area Agrocompetitivo Boletin n°1, Centro de
of the country. The analysis of the botanical origin of these Investigaciones Aplicadas - Universidad Federico Santa
honeys showed the absence of pollen grains of crops and Maria. 2012. p.1-4.
the majority presence of pollen grains of weeds were such
CIMINO, A. M. et al. Effects of neonicotinoid pesticide
as Medicago sativa, Galega officinalis and Brassica rapa,
exposure on human health: A systematic review.
which could be associated with crops controlled by the
Environmental Health Perspectives, 125(2):155-162,
chemicals. Our study raises the concern of neonicotinoids
2017.
residue in Chilean honey samples and the importance of
regular analyses for the detection of residues of pesticides. DECOURTYE, A.; DEVILLERS, J. Ecotoxicity of Neonicotinoid
Although neonicotinoids residue levels were low, they also Insecticides to Bees. In: S. H. THANY. Insect Nicotinic
confirm an actual occurrence of a transfer of neonicotinoid Acetylcholine Receptors. Springer New York, New York,
insecticides from exposed honeybees into honey. NY. p.85-95. 2010.

FAROOQUI, T. A potential link among biogenic amines-

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FIC (IDI 3034723-0) and FIC (IDI 30423322-0) collapse disorder: A unique hypothesis. Neurochemistry
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