Journal of Chromatography B, 990 (2015) 132–140
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of neonicotinoid insecticides and their metabolites in honey bee and honey by liquid chromatography tandem mass spectrometry Malgorzata Gbylik-Sikorska ∗ , Tomasz Sniegocki, Andrzej Posyniak Pharmacology and Toxicology Department, National Veterinary Research Institute (NVRI), al. Partyzantow 57, 24-100 Pulawy, Poland
a r t i c l e
i n f o
Article history: Received 12 January 2015 Accepted 21 March 2015 Available online 1 April 2015 Keywords: Neonicotinoids Metabolites Honey bee Honey LC-MS/MS
a b s t r a c t The original analytical method for the simultaneous determination and confirmation of neonicotinoids insecticides (imidacloprid, clothianidin, acetamiprid, thiametoxam, thiacloprid, nitenpyram, dinotefuran) and some of their metabolites (imidacloprid guanidine, imidacloprid olefin, imidacloprid urea, desnitro-imidacloprid hydrochloride, thiacloprid-amid and acetamiprid-N-desmethyl) in honey bee and honey was developed. Preparation of honey bee samples involves the extraction with mixture of acetonitrile and ethyl acetate followed by cleaned up using the Sep-Pak Alumina N Plus Long cartridges. Honey samples were dissolved in 1% mixture of acetonitrile and ethyl acetate with addition of TEA, then extracts were cleaned up with Strata X-CW cartridges. The identity of analytes was confirmed using liquid chromatography tandem mass spectrometry. All compounds were separated on a Luna C18 column with gradient elution. The whole procedure was validated according to the requirements of SANCO 12571/2013. The average recoveries of the analytes ranged from 85.3% to 112.0%, repeatabilities were in the range of 2.8–11.2%, within-laboratory reproducibility was in the range of 3.3–14.6%, the limits of quantitation were in the range of 0.1–0.5 g kg−1 , depending of analyte and matrices. The validated method was successfully applied for the determination of clothianidin, imidacloprid and imidacloprid urea in real incurred honey bee samples and clothianidin in honey. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The functioning of agriculture and world wild plants without bees is possible, but necessary to this effort is unimaginable, because of the role of insect in the world plants ecosystems as the pollinators is irreplaceable. Moreover bees are one of the most economically important insects because of high production of honey, beeswax, royal jelly, pollen and propolis. All of these products are widely used in food, cosmetic industry, medicine, especially bee venom is increasingly used in alternative medicine—apitherapy. In last few decades it is observed colony collapse disorder (CCD) is still increasing. This state of affairs is influenced by many factors (pollutants, climate changes, parasites, pathogens, plant protection products, especially insecticides). This study focuses specifically on the determination and confirmation of neonicotinoid insecticides and some of their metabolites in honey bee and honey samples, which are one of the most potentially a threat to bee health. Neonicotinoids are one of the synthetic
∗ Corresponding author. Tel.: +48 81 889 31 27; fax: +48 81 886 25 95. E-mail address: [email protected] (M. Gbylik-Sikorska). http://dx.doi.org/10.1016/j.jchromb.2015.03.016 1570-0232/© 2015 Elsevier B.V. All rights reserved.
insecticides which are the fastest growing class in crop protection against sucking insects, moths, butterflies, various species of beetles and other pest herbivores in last 20 years [1,2]. This success is due to the molecular structure (Fig. 1). Commercialised neonicotinoids can be divided into two categories of compounds which have open-chain structure (acetamiprid, clothianidin dinotefuran and nitenpyram) or five/six-member heterocyclic ring structure (imidacloprid, thiacloprid and thiamethoxam) [3]. Neonicotinoids have a part of a molecular structure that is responsible for a particular biological or pharmacological interaction (pharmacophores). In general they are represented by nitro or cyano substitute and also NH, N-Me, S or Me group in their structure (Fig. 1). These types of pharmacophores influenced on neonicotinoids insecticides activity, toxicity and some physicochemical properties which determined the analytical approach [4–6]. The neonicotinoid activity is based on their interaction with the nicotinic acetylocholine receptors (nAChRs), the membrane proteins which are responsible for inducing membrane depolarization in nerve synapses situated in the insect central nervous system (CNS). Neonicotinoids are high selectivity to the insect nAChRs compared to the mammalian nAChRs, which makes them very desirable insecticides in the agricultural industry [1,7,8]. Unfortunately, their main advantage
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Fig. 1. Chemical structure of neonicotinoids and their metabolites, including pharmacophores indication.
proved to be deadly to honey bees and pollinators because of their toxic and neurotoxic properties. The honey bee exposition to neonicotinoids can lead to contamination of apiarian products, especially honey, which is the most commonly consumed bee product and because of the potential threat to human health, the European Union established maximum residue limits (MRLs) for acetamiprid, clothianidin, imidacloprid, thiacloprid and thiamethoxam in the range of 10–200 g kg−1 [9]. Different approach for the neonicotinoids and their metabolites in environmental samples (soil, river water) [10–12], animal tissues [13,14], agricultural (chestnut, shallot, tea, ginger, amaranth, sedum, anion, pepper, lettuce) samples [15–18], alcohol industry products (honey liqueur) [19] and honey bee [20–22] and bees products (honey, bee pollen, beeswax) [22–25] were proposed. These methods used different techniques LC-MS/MS [11,13–16,18–24], GC-MS/MS [17], UPLC-UV [10], LC-h-ED [27], UPLC-DAD [20], CE [28] and LC-amperometric detector [12]). Most of the methods described the determination of several neonicotinoids [10,11,13,14,18,19,25,26,28] or only one neonicotinoid with its metabolites, in different biological matrices [15–17,27]. Only a few methods demonstrated the determination of neonicotinoids or neonicotinoids and their metabolites in honey bee [20,21,23,24] and bee products such as bee pollen [12,22]. However, the reported method for determination neonicotinoids in honey did not include their metabolites. Several sample preparation
techniques, liquid–liquid extraction [23], solid phase extraction [24], QuEChERS [20,22] and combination of them [21], for the sample preparation in honey bee and honey were reported. The presented study reports the development and validation of the analytical method for the determination of 7 neonicotinoids and 6 of their metabolites in honey bee and honey. Proposed method include some of the novelties such as usage of the Sep-Pak Alumina N Plus Long cartridges as the filter for the honey bee supernatants clean-up step and the Strata X-CW cartridges usage for the honey SPE process. To the best of our knowledge and the available literature data, this is the first time that 13 analytes from neonicotinoid group are determined in one analytical protocol.
2. Material and methods 2.1. Reagents All reagents used were of analytical grade, >95% purity. Acetonitrile and ethyl acetate were purchased from J.T. Baker (Deventer, the Netherlands). Triethylamine (TEA) was purchased from Sigma-Aldrich (Steinhiem, Germany). Water was deionised (>18 M cm−1 ) by the Millipore system. Certified standards of neonicotinoids, acetamiprid, acetamiprid-N-desmethyl, clothianidin, dinotefuran, imidacloprid, desnitro imidacloprid
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hydrochloride, nitenpyram, thiacloprid, thiacloprid-amide, thiamethoxam and acetamiprid-d3 , clothianidin-d3 , imidalcoprid-d4 , thiamethoxam-d3 were purchased from Sigma-Aldrich (Steinhiem, Germany). Imidacloprid olefin was purchased from Bayer CropScience (Leverkusen, Germany) and imidacloprid urea, imidacloprid guanidine from Ehrenstorfer GmbH (Augsburg, Germany). Sep-Pak Alumina N Plus Long cartridge were obtained from Waters (Milford, the USA), Strata X-CW (100 mg, 6 mL) cartridges were obtained from Phenomenex (Torrance, CA, the USA), syringe filters 0.22 m PVDF were from Restek (Bellefonte, PA, the USA).
for 15 min, and then centrifuged at 2930 × rcf for 10 min at 1 ◦ C. The whole supernatant was transferred to Strata X-CW cartridge preconditioned with 3 mL of methanol and water. The cartridge was washed up by 5 mL of water, dried under vacuum for 5 min and eluted twice with 3 mL of mixture of acetonitrile and ethyl acetate (8:2, v/v). The eluate was evaporated to dryness under a stream of nitrogen at 45 ± 5 ◦ C. The residue was dissolved in 250 L of water and filtered through 0.22 m PVDF syringe filter into LC vials.
2.2. Standard solutions
The LC-MS/MS analysis was performed on the Agilent 1200 HPLC system (Agilent Technologies, Waldronn, Germany) with an automatic degasser, a binary pump and an auto sampler connected to the AB Sciex QTRAP® 6500 triple quadrupole mass spectrometer (AB Sciex, Framingham, the USA). The chromatographic separation was performed on the Luna C18(2) 100 A column (100 × 2.0 mm, particle size 3 m, Phenomenex, the USA) connected to a C18 pre column 4 mm × 2 mm × 2 m (Phenomenex, the USA), which was maintained at 40 ◦ C. The LC flow was maintained at 300 L min−1 , the injection volume was 5 L. The mobile phase gradient program started at 99% of B then decreases to 10% at 2.0 and 5% at 5 min, held for 2 min. The mobile returned to the initial composition at 7.0 min and equilibrated for another 7 min before the next injection. Electrospray ionisation was performed in the positive mode with the following parameters: resolution Q1 and Q3—unit; ion spray voltage—5500 V, temperature—300 ◦ C, nebuliser gas (N2 )—35; curtain gas (N2 )—20; collision gas (N2 )—16; auxiliary gas—16. The Analyst 1.6.2 software controlled the LC-MS/MS system and processed the data. The data acquisition was in multiple reactions monitoring (MRM) mode. The ion transitions and mass parameters monitored for each analyte are listed in Table 1.
Individual neonicotinoids and their metabolites were prepared at concentration of 1 mg mL−1 in acetonitrile and stored in amber volumetric flasks at −18 ◦ C. The final mixed standard stock solutions were prepared by mixing appropriate volumes of each standard stock solutions of neonicotinoids and their metabolites in deionised water and stored in amber volumetric flasks at 4 ◦ C. Individual internal standard stock solution (IS) were prepared at concentration of 1 mg mL−1 in acetonitrile and stored in amber volumetric flasks at −18 ◦ C. Mixture of four working internal standard solution 1 g mL−1 , (acetamiprid-d3 , clothianidin-d3 , imidalcoprid-d4 and thiamethoxam-d3 ) was prepared in deionised water and stored in amber volumetric flask at 4 ◦ C. It was found that the stability of proven individual stock standard solution of all compounds stored at −18 ◦ C remains unchanged for at least 6 months. The working mixture standard solution was stored at 4 ◦ C and was stable not longer than for 1 months because of the rapid degradation of imidacloprid olefin and imidacloprid urea. 2.3. Sampling procedure For sample preparation step and validation experiments honey bees and honey samples were collected from hives localised on experimental apiary on the laboratory (Swarzedz, Poland). Honey samples were stored at 4 ◦ C until the analysis. Honey bee samples were frozen and stored at −18 ◦ C until the analysis. Before the experiment, samples were, respectively, checked to be free of the analytes. 2.4. Sample preparation 2.4.1. Honey bee Before sample preparation, honey bee was freezed for 10 min in metal vessel with liquid nitrogen followed by homogenisation. An amount of 2 ± 0.05 g of homogenous bee was weighed in a 50 mL centrifuge tube and 40 L of IS mixture was added, sample was mixed and left to incubate at 4 ◦ C in the dark for 30 min. The 8 mL of acetonitrile and ethyl acetate mixture (8:2, v/v) was added, the sample was homogenised with a vortex mixer for 1 min and put in an ultrasonic bath to mix well for 15 min, and then centrifuged at 2930 × rcf for 10 min at 1 ◦ C. The whole of the supernatant was transferred to Sep-Pak Alumina N Plus Long cartridges without preconditioned part (as a filter). The filtered supernatant was collected in glass tube and evaporated to dryness under a stream of nitrogen at 45 ± 5 ◦ C. The residue was dissolved in 300 L of water and filtered through 0.22 m PVDF syringe filter into LC vials. 2.4.2. Honey An amount of 2 ± 0.05 g of honey was weighed in a 50 mL centrifuge tube and 40 L of IS mixture was added, sample was mixed and left to incubate at 4 ◦ C in the dark for 30 min. The 10 mL 1% acetonitrile and ethyl acetate mixture (8:2, v/v) and 200 L of 20% TEA in acetonitrile was added; the sample was homogenised with a vortex mixer for 1 min and put in a ultrasonic bath to mix well at 30 ◦ C
2.5. Liquid chromatography-mass spectrometry
2.6. Method validation The developed method was validated according to the requirements of SANCO 12571/2013 [29]. The linearity, selectivity, repeatability, reproducibility, recovery, the limit of quantitation (LOQ), matrix-effect (ME) and robustness of the method were evaluated. The linearity was performed by the matrix-matched calibration curve which had been prepared by fortifying blank honey bee and honey samples at 8 concentration levels (0.1–500 g kg−1 ) depend of matrix and analyte. The 40 L of IS mixture stock solution (acetamiprid-d3 , clothianidin-d3 , imidacloprid-d4 and thiamethoxam-d3 ) was added to each sample. Quantitative results evaluation was performed by comparing the analyte/internal standard peak area ratio from matrix-matched calibration curve to the analyte/internal standard peak area ratio in analysed samples. Selectivity was checked by analysis of 20 blank honey bee and honey samples collected from different sources which allows to verify the appearance of possible presence of interfering substances around the retention times of the compounds of interest. The repeatability was calculated as the relative standard deviation (RSDr , %) of results obtained after fortifying of six blank honey bee and honey samples at three concentration levels depend of matrix and analyte (Table 2). The spiked samples were prepared and analysed on the same day with the same instrument and the same operators. The within-laboratory reproducibility was calculated as the relative standard deviation (RSDwR , %) of the results obtained after fortifying another two sets of blank samples at the same concentration levels of analysed compounds as for the repeatability and analysing on 2 days with the same instrument and another operators. The average recovery was evaluated in the same experiment as repeatability by comparing the mean measured concentration with the fortified concentration of the samples. The LOQ were estimated as the lowest spike level after analysis of 8
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Table 1 Mass spectrometric parameters for the LC-MS/MS determination of neonicotinoids and their metabolites. Analyte
Retention time (min)
Precursor ion (m/z)
Product ions (m/z)
DP (V)
CE (Ev)
Acetamiprida Acetamiprid-N-desmethyla Clothianidinb Dinotefurana Imidaclopridc Imidacloprid guanidinec Imidacloprid ureac Imidacloprid olefinc Desnitroimidaclopridc Nitenpyrama Thiacloprida Thiacloprid-amida Thiamethoxamd a-Acetamiprid-d3 (IS) b-Clothianidin-d3 (IS) c-Imidacloprid-d4 (IS) d-Thiamethoxam-d3 (IS)
5.67 5.56 5.60 5.35 5.65 5.91 5.48 5.59 5.47 5.38 5.80 5.55 5.50 5.69 5.62 5.67 5.52
223.0 209 249.9 203 256 212 212 254 211 271 253 271 292 226 253 260 295
207.0; 149.0 126.0; 90.0 169.0; 132.0 129.1; 157.1 209.0; 175.0 127.0; 177.0 128.0; 99.0 236.0; 171.0 125.9; 89.9 189.1; 237.1 126.0; 186.0 254.0; 228.0 211.1; 131.8 126.0 172.0 213.0 214.0
30 50 26 31 32 30 30 36 30 29 35 60 35 30 35 30 30
23; 25 25; 40 16; 26 17; 12 20; 25 31; 23 27; 25 13; 27 31; 47 19; 21 25; 24 15; 18 15; 30 23 20 24 17
Table 2 Validation parameters for the LC-MS/MS method for the determination of neonicotinoids and their metabolites. MRL (g kg−1 )
LOQ (g kg−1 )
RSDr a (%)
RSDwR a (%)
Matrix
Spike level (g kg−1 )
Acetamiprid
Honey bee honey
0.1/5/20 0.1/20/50
– 20
0.1 0.1
5.2 4.8
6.8 5.6
−8.1 ± 3.0 −7.3 ± 2.6
112.0 ± 2.1 109.2 ± 1.3
Acetamiprid-N-desmethyl
Honey bee Honey
0.1/5/20 0.1/5/20
– –
0.1 0.1
11.2 10.9
14.6 13.4
−10.4 ± 4.1 −9.2 ± 5.1
85.3 ± 2.0 88.6 ± 2.3
Clothianidin
Honey bee Honey
0.1/5/20 0.1/10/50
– 10
0.1 0.1
6.8 5.3
7.5 5.6
−4.2 ± 2.1 −6.4 ± 4.1
93.2 ± 1.9 101.1 ± 2.0
Dinotefuran
Honey bee Honey
0.1/5/20 0.1/5/20
– –
0.1 0.1
8.9 6.4
11.2 7.1
−6.8 ± 3.2 −5.3 ± 4.5
89.0 ± 1.6 92.2 ± 2.1
Imidacloprid
Honey bee Honey
0.1/5/20 0.1/50/100
– 50
0.1 0.1
3.5 2.8
4.1 3.3
−6.8 ± 4.3 −7.6 ± 5.4
97.8 ± 1.9 98.9 ± 1.3
Imidacloprid guanidine
Honey bee Honey
0.1/5/20 0.1/5/20
– –
0.1 0.1
6.6 5.9
7.2 6.5
−11.2 ± 4.1 12.1 ± 3.8
95.6 ± 2.1 103.2 ± 1.2
Imidacloprid urea
Honey bee Honey
0.1/5/20 0.1/5/20
– –
0.1 0.1
4.6 6.9
5.1 8.2
5.2 ± 4.5 10.5 ± 3.8
97.8 ± 1.6 100.2 ± 2.3
Imidacloprid olefin
Honey bee Honey
0.5/20/50 0.5/20/50
– –
0.5 0.5
7.1 7.6
8.6 9.0
8.4 ± 4.5 −14.5 ± 4.9
108.4 ± 2.1 106.5 ± 2.0
Desnitroimidacloprid
Honey bee Honey
0.5/20/50 0.5/20/50
– –
0.5 0.5
10.1 9.8
12.4 11.6
−16.0 ± 3.8 15.6 ± 4.2
104.2 ± 1.1 101.1 ± 2.7
Nitenpyram
Honey bee Honey
0.1/5/20 0.1/5/20
– –
0.1 0.1
6.4 5.8
7.6 6.7
−6.5 ± 2.6 −7.2 ± 3.4
98.7 ± 1.6 99.3 ± 2.3
Thiacloprid
Honey bee Honey
0.1/5/20 0.1/20/200
– 200
0.1 0.1
4.3 5.6
5.9 9.0
−6.4 ± 4.2 −7.2 ± 3.0
103.1 ± 1.4 106.1 ± 2.1
Thiacloprid-amid
Honey bee Honey
0.5/20/50 0.5/20/50
– –
0.5 0.5
8.3 7.5
9.9 8.9
−9.8 ± 5.6 7.5 ± 4.3
108.5 ± 1.7 105.6 ± 1.8
Thiamethoxam
Honey bee Honey
0.1/5/20 0.1/10/50
– 10
0.1 0.1
5.7 4.3
6.7 8.6
−8.1 ± 5.0 7.8 ± 6.1
99.9 ± 2.4 97.3 ± 1.6
a
Matrix effect (%)
Recovery (%)
Analyte
RSDr and RSDwR was calculated at LOQ level.
fortified blank samples at the minimum detectable concentration level with acceptable accuracy of each analytes. The ME was checked by analysing 3 honey bee and honey samples from different commercial hives, samples were, respectively, checked to be free of the analysed neonicotinoids and their metabolites before the experiment. The ME was evaluated as a percentage of signal intensity of honey bee or honey sample extract fortified after extraction at the 20 g kg−1 concentration level in relation to signal intensity of deionised water fortified at the same concentration level. The robustness of the method was estimated for each analytes of eight fortified blank honey bee and honey samples at the 20 g kg−1 concentration level. Different possible factors that could influence the method results (average recovery and RSDwR ) were checked.
3. Result and discussion 3.1. Optimization of LC-MS/MS parameters The MS settings were optimised with a direct infusion of working standard solutions. According to SANCO 12571/2013 [29], for each compound, one precursor ion and two most abundant product ions were chosen as diagnostic ions. The characteristic mass spectrometric parameters—declustering potential (DP), collision energy (CE), were optimised separately for each analyte (Table 1). The analyses were performed in positive ionisation mode. In order to optimise chromatographic separation, different mobile phases were tested. Most of the reported method used water, acids and
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Fig. 2. LC-MS/MS optimization results for four different chromatographic columns: 1-dinotefuran, 2-nitenpyram, 3-desnitroimidacloprid, 4-imidacloprid urea, 5-thiaclopridamid, 6-thiamethoxam, 7-acetamiprid-N-desmethyl, 8-imidacloprid olefin, 9-clothianidin, 10-imidacloprid, 11-acetamiprid, 12-thiacloprid, 13-imidacloprid guanidine, IS1thiamethoxam-d3 , IS2-clothianidin-d3 , IS3-imidacloprid-d4 , IS4-acetamiprid-d3 .
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Fig. 2. (Continued ).
buffer mobile phases consisting acetic acid, formic acid, ammonium formate as the aqueous phase and acetonitrile or methanol as the organic once [15,20,22–24,27]. In our study, water, acetic acid, ammonium formate, formic acid and their mixtures at different (0.05–1%) concentration levels were checked. The acetonitrile, methanol, ethyl acetate and their combination with additives such as formic acid and ammonium formate were tested as the organic ones. To check which combination of mobile phases that performed the best results, different gradient elution were tested. On the basis of the literature the usage of chromatography column with C18 stationary gave the best result [15,16,19,23,24,26]. In these study, all mobile phases combination were tested coupled with the following HPLC-columns: Luna C18(2) (150 × 2.0 mm, particle size 3 m), Luna C18(2) 100A (100 × 2.0 mm, particle size 3 m), Luna Hilic 200A (50 × 3.0 mm, particle size 3 m), Kinetex C18 100A (100 × 2.1 mm, particle size 2.6 m) and InterSustain C18 (100 × 3.0 mm, particle size 3 m). Between all tested combination, the use of Luna C18(2) 100A (100 × 2.0 mm, particle size 3 m) column with water as the aqueous phase and acetonitrile as the organic ones, enable to achieved the conditions providing high selectivity, short retention time, good peak shape and higher intensity of most compounds (Fig. 2). Also, the adequate internal standards for each analytes were selected at this part of the experiment (Table 1). 3.2. Optimisation of sample preparation The most important part of the developed method was the choice of the extraction mixture to give an acceptable recovery of neonicotinoids and their metabolites from complex matrices such as honey and honey bee. Especially it was challenging to optimise the sample preparation protocol because of different physicochemical properties of analysed compounds. Additionally, it is very important to utilise the clean-up techniques which are efficient enough to eliminate interfering compounds, minimise the loss of analytes before analysis and provide enough sensitivity at the target concentration levels. In the study, some optimisation of extraction procedure and clean-up processes for samples of honey bee and honey was carried out. 3.2.1. Honey bee As it is reported in the literature, acetone, acetonitrile were used as the honey bee sample extraction solvent for the isolation of
matrixes [20–22,27]. At the beginning of the presented experiment, two different ways of extraction procedure: the usage of aqueous and organic solvent as the extractant were checked. At first, water, formic acid, ammonium formate at different concentration levels, coupled with liquid–liquid extraction (LLE) or solid phase extraction (SPE) were tested. Ethyl acetate and dichloromethane at the LLE process and C18, Strata-X and Oasis HLB cartridges at the SPE process were used. Unfortunately, most of this experiment resulted in major losses of some compounds, especially metabolites (imidacloprid guanidine, imidacloprid urea and imidacloprid olefin). In the second experiment, acetonitrile, acetone and ethyl acetate and their mixture at different concentration levels and the following conditions (mechanic shaken, vortex, ultrasonic bath with temperature change) were tested. The acetonitrile and ethyl acetate gave good results as the extraction solvent, and combination in the ratio 8–2 coupled with vortex and ultrasonic bath in room temperature usage, allowed to achieve the best results (best recoveries for all analytes) as it is show at Fig. 3a. Because of the purity of supernatant were not enough (supernatant contain pollen residue, dyes and proteins), it was necessity to use the additional clean-up step. The supernatants clean-up were checked with the usage of different filters (PTFE, Nylon) and cartridges such as Oasis HLB (as a filter) or Sep-Pak Alumina N Plus Long. The best result (good recoveries for all analytes) and reduction of interfering components was achieved with Sep-Pak Alumina N Plus Long (Fig. 3b). In order to further improve the purification of the final extract different filters (PVDF, NYLON syringe filters and Nanosep MF Centrifugal Devices) were tested. The PVDF syringe filters gave the best purity of the extract without any negative influence on recoveries of the analytes.
3.2.2. Honey Honey is one of the most complex matrices of food of animal origin which contained monosaccharides, organic acids, essential oils, dyes, residue of bee pollen and wax. Already, at the beginning it was decided to resign from the use of organic solvents as the extractant because of the insolubility of honey in such as solvents. As the extraction step LLE and SPE were tested in a few experiments [23,24]. In this study, the composition of the water, buffer (ammonium formate) acids (acetic acid and formic acid), with acetonitrile, ethyl acetate, acetone in different concentration levels (0.1–5%), were checked. The usage of aqueous solvent was not sufficient (low recoveries of few analytes), but small addition of organic component significantly influenced for isolation of
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Fig. 3. Recoveries of neonicotinoids and metabolites for a-comparison of organic extractant for honey bee, b-comparison of different supernatant clean-up for honey bee, c-comparison of different SPE cartridges for honey.
analytes from matrix. Good result was obtained with acetonitrile and ethyl acetate mixture as the extraction solvent, but the recovery for some metabolites were lower than 70%. In accordance to SANCO [29], recovery below 70% generally is not accepted. In the previous studies, addition of TEA to extraction or elution solvent to improve isolation of metabolites was used [22,25]. During the experiment, different concentration of TEA in acetonitrile, ethyl acetate were tested. The best result were achieved with 1% mixture of acetonitrile and ethyl acetate (8:2, v/v) in water with addition of 20% TEA in acetonitrile coupled with ultrasonic bath at 30 ◦ C. In order to improve the best clean-up procedure to reduce the presence of impurities, which may lead to the generation of chromatograms with multiple overlapping peaks and a
sloping background, the SPE cartridges were tested. Several SPE sorbents (C18, Strata-X, Strata X-CW and Oasis HLB) were checked for sample clean-up because only this kind of cartridges enables all compounds isolation in neutral conditions. The comparison of analyte recoveries for different SPE cartridges is shown in Fig. 3c. For the elution of the analytes, the suitability of methanol, acetonitrile, ethyl acetate and the combination of them and with TEA were tested. There was no significant impact on metabolites recoveries with the TEA addition to the eluant solvent. The best clean-up conditions were obtained with Strata X-CW cartridges and elution with mixture of acetonitrile and ethyl acetate (8:2, v/v). In case of improving the disposal of interfering substances, the PVDF syringe filters were also checked and applied in procedure.
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Fig. 4. Incurrent samples: a-clothianidin in honey bee—4.0 g kg−1 ), b-clothianidin in honey bee—13.1 g kg−1 , c-clothianidin in honey—13.7 g kg−1 , d-clothianidin in honey—192.8 g kg−1 , e-imidacloprid in honey bee—27.0 g kg−1 , f-imidacloprid urea in honey bee—4.5 g kg−1 .
3.3. Method validation The whole analytical method was validated in accordance to the SANCO 12571/2013, results are provided in Table 2. The matrixmatched curves showed good linearity (r2 > 0.99) for all the analytes over the range LOQ–500 g kg−1 . The concentrations of the analytes were derived directly from the matrix calibration curve with the use of internal standards suitable for each compound (Table 1). The selectivity of the method was found to be satisfactory with no interfering peaks from endogenous compounds in the retention time of the target analytes in honey bee and honey samples. Precision, expressed as the repeatability and within-laboratory reproducibility gave the RSDr and RSDwR values in agreement with the SANCO criteria of RSD ≤ 20%. The RSDr , were in the range of 3.5–11.2% for honey bee and in the range of 2.8–10.9% for honey samples, respectively. The RSDwR , was in the range of 4.1–14.6% for honey bee and 3.3–13.4% for honey samples. Satisfactory average recoveries were calculated in terms of the internal standards.
The average recovery result ranged 85.3–112% for honey bee and 88.6–109.2% for honey samples, respectively and are in accordance with the SANCO validation guideline of recovery, which should be in the range of 70–120%. The LOQ values, with acceptable accuracy of each analytes, are in the range of 0.1–0.5 g kg−1 for honey bee and honey samples. Regarding average ME almost all compounds in honey bee matrices were subjected to ion suppression (4.2–16.0%) with the exception of imidacloprid urea and imidacloprid olefin characterised by low average signal enhancement 5.2% and 8.4%, respectively. For honey samples it was found that the average ME for these matrices were expressed as ion suppression is in the range of 5.3–14.5% but also it was observed significantly more cases of signal enhancement (5 analytes) in the range of 7.5–15.6%. The highest average signal enhancement was calculated for imidacloprid metabolites (10.5–15.6%). The results of the studies indicate that the average ME was observed for all compounds but it is still not affected for determination of them in honey bee and honey samples, because of the average ME ≤ 20%. The signal suppression and
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signal enhancement were minimised by usage of matrix-matched calibration curves and the suitable selected for each compound internal standard. The robustness of the method becomes clear during the optimisation of the optimization of sample preparation. For the robustness estimated for honey bee sample, different volume of the extraction solvent (7 mL, 8 mL and 9 mL), for honey different values of elution mixture (2 × 3 mL, 2 × 3.5 mL, 2 × 4 mL) were tested. In both cases, the ultrasonic bath temperature (25 ◦ C, 30 ◦ C and 35 ◦ C), the temperature of evaporation (40–50 ◦ C) and different size of PVDF syringe filters (0.22 m and 0.45 m) were investigated. There were no significant differences at the evaluation of robustness. In addition, no degradation of the compounds was noted at low and higher temperature ultrasonic bath extraction process. The syringe filter resize and the different evaporation temperatures did not affect average recovery or RSDwR of analytes. 3.4. Real sample application The effectiveness of proposed method was checked by analysing real samples of honey bee and honey obtained courtesy of the Research Institute of Horticulture Apicultural Division in Pulawy. These samples were collected from hives included in the experiment of the oral administration of clothianidin with sugar syrup to honey bee. Another one, honey bee sample came from bee poisoning monitoring with a suspicion of imidacloprid usage and was acquired from National Research Veterinary Institute in Pulawy. For the method quantification of analysed compounds, matrixmatched calibration curves were used. Whereas retention times, comparing analyte/internal standard peak area ratio from matrixmatched calibration curve to analyte/internal standard peak area ratio in analysed samples, were also used for identification. The results of this study indicate that in both honey bee and honey samples from experiment, the presence of clothianidin were confirmed. In honey bees it was found 4.0 g kg−1 and 13.1 g kg−1 of this compound. In honey samples it was found 13.7 g kg−1 and 192.8 g kg−1 of clothianidin, respectively. In honey bee sample from bee poisoning monitoring it was found 27.0 g kg−1 of imidacloprid and 4.5 g kg−1 of imidacloprid urea. All results are shown in Fig. 4. 4. Conclusion A rapid, sensitive invented method, for a fast quantitative and qualitative analysis of 13 analytes from neonicotinoids group and some of their metabolites in honey bee and honey samples has been developed. Both sample preparation procedures are based on the SPE extraction and clean-up subsequently analysed by LC–MS/MS. The proposed method allows to determine all of analysed compounds in a single run protocol. The analyse of real sample shown that developed method can be used as the quantitative and confirmatory analytical method.
Acknowledgement Authors wish to thank Ms Malgorzacie Bienkowskiej the employee of Research Institute of Horticulture Apicultural Division in Pulawy, for the supplying the experimentally incurred by neonicotinoid honey bee and honey samples, also we would like to thanks Andrzejowi Boberowi (NVRI, Pulawy) for the supplying neonicotinoid-free honey bee and honey samples and Tomaszowi Kiljankowi (NVRI, Pulawy), for the supplying the honey bee sample from bee poisoning research. References [1] K. Marsuda, S.D. Buckingham, D. Kleier, J.J. rauh, M. Grauso, D.B. Sattelle, Trends Pharmacol. Sci. 22 (2001) 573–580. [2] J.E. Cresswell, N. Desneux, D. vanEnelsdorp, Pest Manag. Sci. 68 (2012) 819–827. [3] T. Iwasa, N. Motoyama, H.T. Ambrose, R.M. Roe, Crop Protect. 23 (2004) 371–378. [4] M. Tomizawa, J.E. Casida, Ann. Rev. Entomol. 48 (2003) 339–364. [5] J.E. Cresswell, C.J. Page, M.B. Uygun, M. Holmbergh, Y. Li, J.G. Wheeler, I. Laycock, C.J. Pook, N. Hempel de Ibarra, N. Smirnoff, C.R. Tyler, Zoology 115 (2012) 365–371. [6] J.P. van der Sluijs, N.S. Delso, D. Goulson, L. Maxim, J.M. Bonmatin, L.P. Belzunces, Curr. Opin. Environ. Sustainability 5 (2013) 293–305. [7] J. Tan, J.J. Galligan, R.M. Hollingworth, Neuro Toxicol. 28 (2007) 829–842. [8] Y. Wang, J. Cheng, X. Qian, Z. Li, Bioorg. Med. Chem. 15 (2007) 2624–2630. [9] EU Pesticides database http://ec.europa.eu/sanco pesticides/public/?event= activesubstance.selection, accessed: 2014.09.16. [10] F. Sánchez-Bayo, R.V. Hyne, Chemosphere 99 (2014) 143–151. [11] E. Dankyi, C. Gordon, D. Carboo, I.S. Fomsgaard, Sci. Total Environ. 499 (2014) 276–283. [12] N. Ruiz de Erenchun, Z. Gomez de Balugera, M.A. Goicolea, R.J. Barrio, Anal. Chim. Acta 349 (1997) 199–206. [13] Z. Xiao, X. Li, X. Wang, J. Shen, S. Ding, J. Chromatogr. B 879 (2011) 117–122. [14] Z. Xiao, Y. Yang, Y. Li, X. Fan, S. Ding, Anal. Chim. Acta 777 (2013) 32–40. [15] B.M. Kim, J.S. Park, J.H. Choi, A.M.A. El-Aty, T. Wong Na, J.H. Shim, Food Chem. 131 (2012) 1546–1551. [16] T. Yoshida, H. Murakawa, K. Toda, J. Pestic. Sci. 38 (2013) 27–32. [17] A.Y. Ko, Md.M. Rahman, A.M.A. El-Aty, J. Jang, J.H. Park, S.K. Cho, J.H. Shim, Food Chem. 148 (2014) 402–409. [18] W. Xie, C. Han, Y. Qian, H. Ding, X. Chen, J. Xi, J. Chromatogr. A 1218 (2011) 4426–4433. ´ M. Salaˇc, I. Milovanovic, ´ N. [19] P. Jovanov, V. Guzsvány, M. Franko, S. Lazic, ´ Food Res. Int. 55 (2014) 11–19. Nedeljkovic, [20] A. Tapparro, C. Giorio, L. Soldà, S. Bigialli, D. Marton, M. Marzaro, V. Girolami, Anal. Bioanal. Chem. 405 (2013) 1007–1014. [21] A.C. Martel, C. Lair, Int. J. Environ. Anal. Chem. 91 (2011) 978–988. [22] A. Kamel, J. Agric. Food Chem. 58 (2010) 5926–5931. ˇ c, ´ M. Sakaˇc, B. Sari ´ V. Banjac, Talanta [23] P. Jovanov, V. Guzsvány, M. Franko, S. Lazic, 111 (2013) 125–133. [24] P. Fidente, S. Seccia, F. Vanni, P. Morrica, J. Chromatogr. A 1094 (2005) 175–178. [25] B. Giroud, A. Vauchez, E. Vulliet, L. Wiest, A. Buleté, J. Chromatogr. A 1316 (2013) 53–61. [26] K.P. Yánez, J.L. Bernal, M.J. Nozal, M.T. Martin, J. Bernal, J. Chromatogr. A 1285 (2013) 110–117. [27] M. Rancan, A.G. Sabatini, G. Achilli, G.C. Galletti, Anal. Chim. Acta 555 (2006) 20–24. [28] L. Sánchez-Hernández, D. Hernández-Dominquez, J. Bernal, C. Neusüß, M.T. Martin, J.L. Bernal, J. Chromatogr. A 1359 (2014) 317–324. [29] Anon, SANCO/12571/2013—Guidance document on analytical quality control and validation procedures for pesticide residues analysis in food and feed, 19 November 2013 rev. 0.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of neonicotinoid insecticides and their metabolites in honey bee and honey by liquid chromatography tandem mass spectrometry Malgorzata Gbylik-Sikorska ∗ , Tomasz Sniegocki, Andrzej Posyniak Pharmacology and Toxicology Department, National Veterinary Research Institute (NVRI), al. Partyzantow 57, 24-100 Pulawy, Poland
a r t i c l e
i n f o
Article history: Received 12 January 2015 Accepted 21 March 2015 Available online 1 April 2015 Keywords: Neonicotinoids Metabolites Honey bee Honey LC-MS/MS
a b s t r a c t The original analytical method for the simultaneous determination and confirmation of neonicotinoids insecticides (imidacloprid, clothianidin, acetamiprid, thiametoxam, thiacloprid, nitenpyram, dinotefuran) and some of their metabolites (imidacloprid guanidine, imidacloprid olefin, imidacloprid urea, desnitro-imidacloprid hydrochloride, thiacloprid-amid and acetamiprid-N-desmethyl) in honey bee and honey was developed. Preparation of honey bee samples involves the extraction with mixture of acetonitrile and ethyl acetate followed by cleaned up using the Sep-Pak Alumina N Plus Long cartridges. Honey samples were dissolved in 1% mixture of acetonitrile and ethyl acetate with addition of TEA, then extracts were cleaned up with Strata X-CW cartridges. The identity of analytes was confirmed using liquid chromatography tandem mass spectrometry. All compounds were separated on a Luna C18 column with gradient elution. The whole procedure was validated according to the requirements of SANCO 12571/2013. The average recoveries of the analytes ranged from 85.3% to 112.0%, repeatabilities were in the range of 2.8–11.2%, within-laboratory reproducibility was in the range of 3.3–14.6%, the limits of quantitation were in the range of 0.1–0.5 g kg−1 , depending of analyte and matrices. The validated method was successfully applied for the determination of clothianidin, imidacloprid and imidacloprid urea in real incurred honey bee samples and clothianidin in honey. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The functioning of agriculture and world wild plants without bees is possible, but necessary to this effort is unimaginable, because of the role of insect in the world plants ecosystems as the pollinators is irreplaceable. Moreover bees are one of the most economically important insects because of high production of honey, beeswax, royal jelly, pollen and propolis. All of these products are widely used in food, cosmetic industry, medicine, especially bee venom is increasingly used in alternative medicine—apitherapy. In last few decades it is observed colony collapse disorder (CCD) is still increasing. This state of affairs is influenced by many factors (pollutants, climate changes, parasites, pathogens, plant protection products, especially insecticides). This study focuses specifically on the determination and confirmation of neonicotinoid insecticides and some of their metabolites in honey bee and honey samples, which are one of the most potentially a threat to bee health. Neonicotinoids are one of the synthetic
∗ Corresponding author. Tel.: +48 81 889 31 27; fax: +48 81 886 25 95. E-mail address: [email protected] (M. Gbylik-Sikorska). http://dx.doi.org/10.1016/j.jchromb.2015.03.016 1570-0232/© 2015 Elsevier B.V. All rights reserved.
insecticides which are the fastest growing class in crop protection against sucking insects, moths, butterflies, various species of beetles and other pest herbivores in last 20 years [1,2]. This success is due to the molecular structure (Fig. 1). Commercialised neonicotinoids can be divided into two categories of compounds which have open-chain structure (acetamiprid, clothianidin dinotefuran and nitenpyram) or five/six-member heterocyclic ring structure (imidacloprid, thiacloprid and thiamethoxam) [3]. Neonicotinoids have a part of a molecular structure that is responsible for a particular biological or pharmacological interaction (pharmacophores). In general they are represented by nitro or cyano substitute and also NH, N-Me, S or Me group in their structure (Fig. 1). These types of pharmacophores influenced on neonicotinoids insecticides activity, toxicity and some physicochemical properties which determined the analytical approach [4–6]. The neonicotinoid activity is based on their interaction with the nicotinic acetylocholine receptors (nAChRs), the membrane proteins which are responsible for inducing membrane depolarization in nerve synapses situated in the insect central nervous system (CNS). Neonicotinoids are high selectivity to the insect nAChRs compared to the mammalian nAChRs, which makes them very desirable insecticides in the agricultural industry [1,7,8]. Unfortunately, their main advantage
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Fig. 1. Chemical structure of neonicotinoids and their metabolites, including pharmacophores indication.
proved to be deadly to honey bees and pollinators because of their toxic and neurotoxic properties. The honey bee exposition to neonicotinoids can lead to contamination of apiarian products, especially honey, which is the most commonly consumed bee product and because of the potential threat to human health, the European Union established maximum residue limits (MRLs) for acetamiprid, clothianidin, imidacloprid, thiacloprid and thiamethoxam in the range of 10–200 g kg−1 [9]. Different approach for the neonicotinoids and their metabolites in environmental samples (soil, river water) [10–12], animal tissues [13,14], agricultural (chestnut, shallot, tea, ginger, amaranth, sedum, anion, pepper, lettuce) samples [15–18], alcohol industry products (honey liqueur) [19] and honey bee [20–22] and bees products (honey, bee pollen, beeswax) [22–25] were proposed. These methods used different techniques LC-MS/MS [11,13–16,18–24], GC-MS/MS [17], UPLC-UV [10], LC-h-ED [27], UPLC-DAD [20], CE [28] and LC-amperometric detector [12]). Most of the methods described the determination of several neonicotinoids [10,11,13,14,18,19,25,26,28] or only one neonicotinoid with its metabolites, in different biological matrices [15–17,27]. Only a few methods demonstrated the determination of neonicotinoids or neonicotinoids and their metabolites in honey bee [20,21,23,24] and bee products such as bee pollen [12,22]. However, the reported method for determination neonicotinoids in honey did not include their metabolites. Several sample preparation
techniques, liquid–liquid extraction [23], solid phase extraction [24], QuEChERS [20,22] and combination of them [21], for the sample preparation in honey bee and honey were reported. The presented study reports the development and validation of the analytical method for the determination of 7 neonicotinoids and 6 of their metabolites in honey bee and honey. Proposed method include some of the novelties such as usage of the Sep-Pak Alumina N Plus Long cartridges as the filter for the honey bee supernatants clean-up step and the Strata X-CW cartridges usage for the honey SPE process. To the best of our knowledge and the available literature data, this is the first time that 13 analytes from neonicotinoid group are determined in one analytical protocol.
2. Material and methods 2.1. Reagents All reagents used were of analytical grade, >95% purity. Acetonitrile and ethyl acetate were purchased from J.T. Baker (Deventer, the Netherlands). Triethylamine (TEA) was purchased from Sigma-Aldrich (Steinhiem, Germany). Water was deionised (>18 M cm−1 ) by the Millipore system. Certified standards of neonicotinoids, acetamiprid, acetamiprid-N-desmethyl, clothianidin, dinotefuran, imidacloprid, desnitro imidacloprid
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hydrochloride, nitenpyram, thiacloprid, thiacloprid-amide, thiamethoxam and acetamiprid-d3 , clothianidin-d3 , imidalcoprid-d4 , thiamethoxam-d3 were purchased from Sigma-Aldrich (Steinhiem, Germany). Imidacloprid olefin was purchased from Bayer CropScience (Leverkusen, Germany) and imidacloprid urea, imidacloprid guanidine from Ehrenstorfer GmbH (Augsburg, Germany). Sep-Pak Alumina N Plus Long cartridge were obtained from Waters (Milford, the USA), Strata X-CW (100 mg, 6 mL) cartridges were obtained from Phenomenex (Torrance, CA, the USA), syringe filters 0.22 m PVDF were from Restek (Bellefonte, PA, the USA).
for 15 min, and then centrifuged at 2930 × rcf for 10 min at 1 ◦ C. The whole supernatant was transferred to Strata X-CW cartridge preconditioned with 3 mL of methanol and water. The cartridge was washed up by 5 mL of water, dried under vacuum for 5 min and eluted twice with 3 mL of mixture of acetonitrile and ethyl acetate (8:2, v/v). The eluate was evaporated to dryness under a stream of nitrogen at 45 ± 5 ◦ C. The residue was dissolved in 250 L of water and filtered through 0.22 m PVDF syringe filter into LC vials.
2.2. Standard solutions
The LC-MS/MS analysis was performed on the Agilent 1200 HPLC system (Agilent Technologies, Waldronn, Germany) with an automatic degasser, a binary pump and an auto sampler connected to the AB Sciex QTRAP® 6500 triple quadrupole mass spectrometer (AB Sciex, Framingham, the USA). The chromatographic separation was performed on the Luna C18(2) 100 A column (100 × 2.0 mm, particle size 3 m, Phenomenex, the USA) connected to a C18 pre column 4 mm × 2 mm × 2 m (Phenomenex, the USA), which was maintained at 40 ◦ C. The LC flow was maintained at 300 L min−1 , the injection volume was 5 L. The mobile phase gradient program started at 99% of B then decreases to 10% at 2.0 and 5% at 5 min, held for 2 min. The mobile returned to the initial composition at 7.0 min and equilibrated for another 7 min before the next injection. Electrospray ionisation was performed in the positive mode with the following parameters: resolution Q1 and Q3—unit; ion spray voltage—5500 V, temperature—300 ◦ C, nebuliser gas (N2 )—35; curtain gas (N2 )—20; collision gas (N2 )—16; auxiliary gas—16. The Analyst 1.6.2 software controlled the LC-MS/MS system and processed the data. The data acquisition was in multiple reactions monitoring (MRM) mode. The ion transitions and mass parameters monitored for each analyte are listed in Table 1.
Individual neonicotinoids and their metabolites were prepared at concentration of 1 mg mL−1 in acetonitrile and stored in amber volumetric flasks at −18 ◦ C. The final mixed standard stock solutions were prepared by mixing appropriate volumes of each standard stock solutions of neonicotinoids and their metabolites in deionised water and stored in amber volumetric flasks at 4 ◦ C. Individual internal standard stock solution (IS) were prepared at concentration of 1 mg mL−1 in acetonitrile and stored in amber volumetric flasks at −18 ◦ C. Mixture of four working internal standard solution 1 g mL−1 , (acetamiprid-d3 , clothianidin-d3 , imidalcoprid-d4 and thiamethoxam-d3 ) was prepared in deionised water and stored in amber volumetric flask at 4 ◦ C. It was found that the stability of proven individual stock standard solution of all compounds stored at −18 ◦ C remains unchanged for at least 6 months. The working mixture standard solution was stored at 4 ◦ C and was stable not longer than for 1 months because of the rapid degradation of imidacloprid olefin and imidacloprid urea. 2.3. Sampling procedure For sample preparation step and validation experiments honey bees and honey samples were collected from hives localised on experimental apiary on the laboratory (Swarzedz, Poland). Honey samples were stored at 4 ◦ C until the analysis. Honey bee samples were frozen and stored at −18 ◦ C until the analysis. Before the experiment, samples were, respectively, checked to be free of the analytes. 2.4. Sample preparation 2.4.1. Honey bee Before sample preparation, honey bee was freezed for 10 min in metal vessel with liquid nitrogen followed by homogenisation. An amount of 2 ± 0.05 g of homogenous bee was weighed in a 50 mL centrifuge tube and 40 L of IS mixture was added, sample was mixed and left to incubate at 4 ◦ C in the dark for 30 min. The 8 mL of acetonitrile and ethyl acetate mixture (8:2, v/v) was added, the sample was homogenised with a vortex mixer for 1 min and put in an ultrasonic bath to mix well for 15 min, and then centrifuged at 2930 × rcf for 10 min at 1 ◦ C. The whole of the supernatant was transferred to Sep-Pak Alumina N Plus Long cartridges without preconditioned part (as a filter). The filtered supernatant was collected in glass tube and evaporated to dryness under a stream of nitrogen at 45 ± 5 ◦ C. The residue was dissolved in 300 L of water and filtered through 0.22 m PVDF syringe filter into LC vials. 2.4.2. Honey An amount of 2 ± 0.05 g of honey was weighed in a 50 mL centrifuge tube and 40 L of IS mixture was added, sample was mixed and left to incubate at 4 ◦ C in the dark for 30 min. The 10 mL 1% acetonitrile and ethyl acetate mixture (8:2, v/v) and 200 L of 20% TEA in acetonitrile was added; the sample was homogenised with a vortex mixer for 1 min and put in a ultrasonic bath to mix well at 30 ◦ C
2.5. Liquid chromatography-mass spectrometry
2.6. Method validation The developed method was validated according to the requirements of SANCO 12571/2013 [29]. The linearity, selectivity, repeatability, reproducibility, recovery, the limit of quantitation (LOQ), matrix-effect (ME) and robustness of the method were evaluated. The linearity was performed by the matrix-matched calibration curve which had been prepared by fortifying blank honey bee and honey samples at 8 concentration levels (0.1–500 g kg−1 ) depend of matrix and analyte. The 40 L of IS mixture stock solution (acetamiprid-d3 , clothianidin-d3 , imidacloprid-d4 and thiamethoxam-d3 ) was added to each sample. Quantitative results evaluation was performed by comparing the analyte/internal standard peak area ratio from matrix-matched calibration curve to the analyte/internal standard peak area ratio in analysed samples. Selectivity was checked by analysis of 20 blank honey bee and honey samples collected from different sources which allows to verify the appearance of possible presence of interfering substances around the retention times of the compounds of interest. The repeatability was calculated as the relative standard deviation (RSDr , %) of results obtained after fortifying of six blank honey bee and honey samples at three concentration levels depend of matrix and analyte (Table 2). The spiked samples were prepared and analysed on the same day with the same instrument and the same operators. The within-laboratory reproducibility was calculated as the relative standard deviation (RSDwR , %) of the results obtained after fortifying another two sets of blank samples at the same concentration levels of analysed compounds as for the repeatability and analysing on 2 days with the same instrument and another operators. The average recovery was evaluated in the same experiment as repeatability by comparing the mean measured concentration with the fortified concentration of the samples. The LOQ were estimated as the lowest spike level after analysis of 8
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Table 1 Mass spectrometric parameters for the LC-MS/MS determination of neonicotinoids and their metabolites. Analyte
Retention time (min)
Precursor ion (m/z)
Product ions (m/z)
DP (V)
CE (Ev)
Acetamiprida Acetamiprid-N-desmethyla Clothianidinb Dinotefurana Imidaclopridc Imidacloprid guanidinec Imidacloprid ureac Imidacloprid olefinc Desnitroimidaclopridc Nitenpyrama Thiacloprida Thiacloprid-amida Thiamethoxamd a-Acetamiprid-d3 (IS) b-Clothianidin-d3 (IS) c-Imidacloprid-d4 (IS) d-Thiamethoxam-d3 (IS)
5.67 5.56 5.60 5.35 5.65 5.91 5.48 5.59 5.47 5.38 5.80 5.55 5.50 5.69 5.62 5.67 5.52
223.0 209 249.9 203 256 212 212 254 211 271 253 271 292 226 253 260 295
207.0; 149.0 126.0; 90.0 169.0; 132.0 129.1; 157.1 209.0; 175.0 127.0; 177.0 128.0; 99.0 236.0; 171.0 125.9; 89.9 189.1; 237.1 126.0; 186.0 254.0; 228.0 211.1; 131.8 126.0 172.0 213.0 214.0
30 50 26 31 32 30 30 36 30 29 35 60 35 30 35 30 30
23; 25 25; 40 16; 26 17; 12 20; 25 31; 23 27; 25 13; 27 31; 47 19; 21 25; 24 15; 18 15; 30 23 20 24 17
Table 2 Validation parameters for the LC-MS/MS method for the determination of neonicotinoids and their metabolites. MRL (g kg−1 )
LOQ (g kg−1 )
RSDr a (%)
RSDwR a (%)
Matrix
Spike level (g kg−1 )
Acetamiprid
Honey bee honey
0.1/5/20 0.1/20/50
– 20
0.1 0.1
5.2 4.8
6.8 5.6
−8.1 ± 3.0 −7.3 ± 2.6
112.0 ± 2.1 109.2 ± 1.3
Acetamiprid-N-desmethyl
Honey bee Honey
0.1/5/20 0.1/5/20
– –
0.1 0.1
11.2 10.9
14.6 13.4
−10.4 ± 4.1 −9.2 ± 5.1
85.3 ± 2.0 88.6 ± 2.3
Clothianidin
Honey bee Honey
0.1/5/20 0.1/10/50
– 10
0.1 0.1
6.8 5.3
7.5 5.6
−4.2 ± 2.1 −6.4 ± 4.1
93.2 ± 1.9 101.1 ± 2.0
Dinotefuran
Honey bee Honey
0.1/5/20 0.1/5/20
– –
0.1 0.1
8.9 6.4
11.2 7.1
−6.8 ± 3.2 −5.3 ± 4.5
89.0 ± 1.6 92.2 ± 2.1
Imidacloprid
Honey bee Honey
0.1/5/20 0.1/50/100
– 50
0.1 0.1
3.5 2.8
4.1 3.3
−6.8 ± 4.3 −7.6 ± 5.4
97.8 ± 1.9 98.9 ± 1.3
Imidacloprid guanidine
Honey bee Honey
0.1/5/20 0.1/5/20
– –
0.1 0.1
6.6 5.9
7.2 6.5
−11.2 ± 4.1 12.1 ± 3.8
95.6 ± 2.1 103.2 ± 1.2
Imidacloprid urea
Honey bee Honey
0.1/5/20 0.1/5/20
– –
0.1 0.1
4.6 6.9
5.1 8.2
5.2 ± 4.5 10.5 ± 3.8
97.8 ± 1.6 100.2 ± 2.3
Imidacloprid olefin
Honey bee Honey
0.5/20/50 0.5/20/50
– –
0.5 0.5
7.1 7.6
8.6 9.0
8.4 ± 4.5 −14.5 ± 4.9
108.4 ± 2.1 106.5 ± 2.0
Desnitroimidacloprid
Honey bee Honey
0.5/20/50 0.5/20/50
– –
0.5 0.5
10.1 9.8
12.4 11.6
−16.0 ± 3.8 15.6 ± 4.2
104.2 ± 1.1 101.1 ± 2.7
Nitenpyram
Honey bee Honey
0.1/5/20 0.1/5/20
– –
0.1 0.1
6.4 5.8
7.6 6.7
−6.5 ± 2.6 −7.2 ± 3.4
98.7 ± 1.6 99.3 ± 2.3
Thiacloprid
Honey bee Honey
0.1/5/20 0.1/20/200
– 200
0.1 0.1
4.3 5.6
5.9 9.0
−6.4 ± 4.2 −7.2 ± 3.0
103.1 ± 1.4 106.1 ± 2.1
Thiacloprid-amid
Honey bee Honey
0.5/20/50 0.5/20/50
– –
0.5 0.5
8.3 7.5
9.9 8.9
−9.8 ± 5.6 7.5 ± 4.3
108.5 ± 1.7 105.6 ± 1.8
Thiamethoxam
Honey bee Honey
0.1/5/20 0.1/10/50
– 10
0.1 0.1
5.7 4.3
6.7 8.6
−8.1 ± 5.0 7.8 ± 6.1
99.9 ± 2.4 97.3 ± 1.6
a
Matrix effect (%)
Recovery (%)
Analyte
RSDr and RSDwR was calculated at LOQ level.
fortified blank samples at the minimum detectable concentration level with acceptable accuracy of each analytes. The ME was checked by analysing 3 honey bee and honey samples from different commercial hives, samples were, respectively, checked to be free of the analysed neonicotinoids and their metabolites before the experiment. The ME was evaluated as a percentage of signal intensity of honey bee or honey sample extract fortified after extraction at the 20 g kg−1 concentration level in relation to signal intensity of deionised water fortified at the same concentration level. The robustness of the method was estimated for each analytes of eight fortified blank honey bee and honey samples at the 20 g kg−1 concentration level. Different possible factors that could influence the method results (average recovery and RSDwR ) were checked.
3. Result and discussion 3.1. Optimization of LC-MS/MS parameters The MS settings were optimised with a direct infusion of working standard solutions. According to SANCO 12571/2013 [29], for each compound, one precursor ion and two most abundant product ions were chosen as diagnostic ions. The characteristic mass spectrometric parameters—declustering potential (DP), collision energy (CE), were optimised separately for each analyte (Table 1). The analyses were performed in positive ionisation mode. In order to optimise chromatographic separation, different mobile phases were tested. Most of the reported method used water, acids and
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Fig. 2. LC-MS/MS optimization results for four different chromatographic columns: 1-dinotefuran, 2-nitenpyram, 3-desnitroimidacloprid, 4-imidacloprid urea, 5-thiaclopridamid, 6-thiamethoxam, 7-acetamiprid-N-desmethyl, 8-imidacloprid olefin, 9-clothianidin, 10-imidacloprid, 11-acetamiprid, 12-thiacloprid, 13-imidacloprid guanidine, IS1thiamethoxam-d3 , IS2-clothianidin-d3 , IS3-imidacloprid-d4 , IS4-acetamiprid-d3 .
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Fig. 2. (Continued ).
buffer mobile phases consisting acetic acid, formic acid, ammonium formate as the aqueous phase and acetonitrile or methanol as the organic once [15,20,22–24,27]. In our study, water, acetic acid, ammonium formate, formic acid and their mixtures at different (0.05–1%) concentration levels were checked. The acetonitrile, methanol, ethyl acetate and their combination with additives such as formic acid and ammonium formate were tested as the organic ones. To check which combination of mobile phases that performed the best results, different gradient elution were tested. On the basis of the literature the usage of chromatography column with C18 stationary gave the best result [15,16,19,23,24,26]. In these study, all mobile phases combination were tested coupled with the following HPLC-columns: Luna C18(2) (150 × 2.0 mm, particle size 3 m), Luna C18(2) 100A (100 × 2.0 mm, particle size 3 m), Luna Hilic 200A (50 × 3.0 mm, particle size 3 m), Kinetex C18 100A (100 × 2.1 mm, particle size 2.6 m) and InterSustain C18 (100 × 3.0 mm, particle size 3 m). Between all tested combination, the use of Luna C18(2) 100A (100 × 2.0 mm, particle size 3 m) column with water as the aqueous phase and acetonitrile as the organic ones, enable to achieved the conditions providing high selectivity, short retention time, good peak shape and higher intensity of most compounds (Fig. 2). Also, the adequate internal standards for each analytes were selected at this part of the experiment (Table 1). 3.2. Optimisation of sample preparation The most important part of the developed method was the choice of the extraction mixture to give an acceptable recovery of neonicotinoids and their metabolites from complex matrices such as honey and honey bee. Especially it was challenging to optimise the sample preparation protocol because of different physicochemical properties of analysed compounds. Additionally, it is very important to utilise the clean-up techniques which are efficient enough to eliminate interfering compounds, minimise the loss of analytes before analysis and provide enough sensitivity at the target concentration levels. In the study, some optimisation of extraction procedure and clean-up processes for samples of honey bee and honey was carried out. 3.2.1. Honey bee As it is reported in the literature, acetone, acetonitrile were used as the honey bee sample extraction solvent for the isolation of
matrixes [20–22,27]. At the beginning of the presented experiment, two different ways of extraction procedure: the usage of aqueous and organic solvent as the extractant were checked. At first, water, formic acid, ammonium formate at different concentration levels, coupled with liquid–liquid extraction (LLE) or solid phase extraction (SPE) were tested. Ethyl acetate and dichloromethane at the LLE process and C18, Strata-X and Oasis HLB cartridges at the SPE process were used. Unfortunately, most of this experiment resulted in major losses of some compounds, especially metabolites (imidacloprid guanidine, imidacloprid urea and imidacloprid olefin). In the second experiment, acetonitrile, acetone and ethyl acetate and their mixture at different concentration levels and the following conditions (mechanic shaken, vortex, ultrasonic bath with temperature change) were tested. The acetonitrile and ethyl acetate gave good results as the extraction solvent, and combination in the ratio 8–2 coupled with vortex and ultrasonic bath in room temperature usage, allowed to achieve the best results (best recoveries for all analytes) as it is show at Fig. 3a. Because of the purity of supernatant were not enough (supernatant contain pollen residue, dyes and proteins), it was necessity to use the additional clean-up step. The supernatants clean-up were checked with the usage of different filters (PTFE, Nylon) and cartridges such as Oasis HLB (as a filter) or Sep-Pak Alumina N Plus Long. The best result (good recoveries for all analytes) and reduction of interfering components was achieved with Sep-Pak Alumina N Plus Long (Fig. 3b). In order to further improve the purification of the final extract different filters (PVDF, NYLON syringe filters and Nanosep MF Centrifugal Devices) were tested. The PVDF syringe filters gave the best purity of the extract without any negative influence on recoveries of the analytes.
3.2.2. Honey Honey is one of the most complex matrices of food of animal origin which contained monosaccharides, organic acids, essential oils, dyes, residue of bee pollen and wax. Already, at the beginning it was decided to resign from the use of organic solvents as the extractant because of the insolubility of honey in such as solvents. As the extraction step LLE and SPE were tested in a few experiments [23,24]. In this study, the composition of the water, buffer (ammonium formate) acids (acetic acid and formic acid), with acetonitrile, ethyl acetate, acetone in different concentration levels (0.1–5%), were checked. The usage of aqueous solvent was not sufficient (low recoveries of few analytes), but small addition of organic component significantly influenced for isolation of
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Fig. 3. Recoveries of neonicotinoids and metabolites for a-comparison of organic extractant for honey bee, b-comparison of different supernatant clean-up for honey bee, c-comparison of different SPE cartridges for honey.
analytes from matrix. Good result was obtained with acetonitrile and ethyl acetate mixture as the extraction solvent, but the recovery for some metabolites were lower than 70%. In accordance to SANCO [29], recovery below 70% generally is not accepted. In the previous studies, addition of TEA to extraction or elution solvent to improve isolation of metabolites was used [22,25]. During the experiment, different concentration of TEA in acetonitrile, ethyl acetate were tested. The best result were achieved with 1% mixture of acetonitrile and ethyl acetate (8:2, v/v) in water with addition of 20% TEA in acetonitrile coupled with ultrasonic bath at 30 ◦ C. In order to improve the best clean-up procedure to reduce the presence of impurities, which may lead to the generation of chromatograms with multiple overlapping peaks and a
sloping background, the SPE cartridges were tested. Several SPE sorbents (C18, Strata-X, Strata X-CW and Oasis HLB) were checked for sample clean-up because only this kind of cartridges enables all compounds isolation in neutral conditions. The comparison of analyte recoveries for different SPE cartridges is shown in Fig. 3c. For the elution of the analytes, the suitability of methanol, acetonitrile, ethyl acetate and the combination of them and with TEA were tested. There was no significant impact on metabolites recoveries with the TEA addition to the eluant solvent. The best clean-up conditions were obtained with Strata X-CW cartridges and elution with mixture of acetonitrile and ethyl acetate (8:2, v/v). In case of improving the disposal of interfering substances, the PVDF syringe filters were also checked and applied in procedure.
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Fig. 4. Incurrent samples: a-clothianidin in honey bee—4.0 g kg−1 ), b-clothianidin in honey bee—13.1 g kg−1 , c-clothianidin in honey—13.7 g kg−1 , d-clothianidin in honey—192.8 g kg−1 , e-imidacloprid in honey bee—27.0 g kg−1 , f-imidacloprid urea in honey bee—4.5 g kg−1 .
3.3. Method validation The whole analytical method was validated in accordance to the SANCO 12571/2013, results are provided in Table 2. The matrixmatched curves showed good linearity (r2 > 0.99) for all the analytes over the range LOQ–500 g kg−1 . The concentrations of the analytes were derived directly from the matrix calibration curve with the use of internal standards suitable for each compound (Table 1). The selectivity of the method was found to be satisfactory with no interfering peaks from endogenous compounds in the retention time of the target analytes in honey bee and honey samples. Precision, expressed as the repeatability and within-laboratory reproducibility gave the RSDr and RSDwR values in agreement with the SANCO criteria of RSD ≤ 20%. The RSDr , were in the range of 3.5–11.2% for honey bee and in the range of 2.8–10.9% for honey samples, respectively. The RSDwR , was in the range of 4.1–14.6% for honey bee and 3.3–13.4% for honey samples. Satisfactory average recoveries were calculated in terms of the internal standards.
The average recovery result ranged 85.3–112% for honey bee and 88.6–109.2% for honey samples, respectively and are in accordance with the SANCO validation guideline of recovery, which should be in the range of 70–120%. The LOQ values, with acceptable accuracy of each analytes, are in the range of 0.1–0.5 g kg−1 for honey bee and honey samples. Regarding average ME almost all compounds in honey bee matrices were subjected to ion suppression (4.2–16.0%) with the exception of imidacloprid urea and imidacloprid olefin characterised by low average signal enhancement 5.2% and 8.4%, respectively. For honey samples it was found that the average ME for these matrices were expressed as ion suppression is in the range of 5.3–14.5% but also it was observed significantly more cases of signal enhancement (5 analytes) in the range of 7.5–15.6%. The highest average signal enhancement was calculated for imidacloprid metabolites (10.5–15.6%). The results of the studies indicate that the average ME was observed for all compounds but it is still not affected for determination of them in honey bee and honey samples, because of the average ME ≤ 20%. The signal suppression and
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signal enhancement were minimised by usage of matrix-matched calibration curves and the suitable selected for each compound internal standard. The robustness of the method becomes clear during the optimisation of the optimization of sample preparation. For the robustness estimated for honey bee sample, different volume of the extraction solvent (7 mL, 8 mL and 9 mL), for honey different values of elution mixture (2 × 3 mL, 2 × 3.5 mL, 2 × 4 mL) were tested. In both cases, the ultrasonic bath temperature (25 ◦ C, 30 ◦ C and 35 ◦ C), the temperature of evaporation (40–50 ◦ C) and different size of PVDF syringe filters (0.22 m and 0.45 m) were investigated. There were no significant differences at the evaluation of robustness. In addition, no degradation of the compounds was noted at low and higher temperature ultrasonic bath extraction process. The syringe filter resize and the different evaporation temperatures did not affect average recovery or RSDwR of analytes. 3.4. Real sample application The effectiveness of proposed method was checked by analysing real samples of honey bee and honey obtained courtesy of the Research Institute of Horticulture Apicultural Division in Pulawy. These samples were collected from hives included in the experiment of the oral administration of clothianidin with sugar syrup to honey bee. Another one, honey bee sample came from bee poisoning monitoring with a suspicion of imidacloprid usage and was acquired from National Research Veterinary Institute in Pulawy. For the method quantification of analysed compounds, matrixmatched calibration curves were used. Whereas retention times, comparing analyte/internal standard peak area ratio from matrixmatched calibration curve to analyte/internal standard peak area ratio in analysed samples, were also used for identification. The results of this study indicate that in both honey bee and honey samples from experiment, the presence of clothianidin were confirmed. In honey bees it was found 4.0 g kg−1 and 13.1 g kg−1 of this compound. In honey samples it was found 13.7 g kg−1 and 192.8 g kg−1 of clothianidin, respectively. In honey bee sample from bee poisoning monitoring it was found 27.0 g kg−1 of imidacloprid and 4.5 g kg−1 of imidacloprid urea. All results are shown in Fig. 4. 4. Conclusion A rapid, sensitive invented method, for a fast quantitative and qualitative analysis of 13 analytes from neonicotinoids group and some of their metabolites in honey bee and honey samples has been developed. Both sample preparation procedures are based on the SPE extraction and clean-up subsequently analysed by LC–MS/MS. The proposed method allows to determine all of analysed compounds in a single run protocol. The analyse of real sample shown that developed method can be used as the quantitative and confirmatory analytical method.
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