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Article

Optimization of Method for Pesticide Detection in Honey by Using Liquid and Gas Chromatography Coupled with Mass Spectrometric Detection

by
Mariana O. Almeida
1,
Silvia Catarina S. Oloris
1,
Vanessa Heloisa F. Faria
1,
Márcia Cassimira M. Ribeiro
1,
Daniel M. Cantini
1 and
Benito Soto-Blanco
2,*
1
Instituto Otávio Magalhães, Fundação Ezequiel Dias (Funed), Rua Conde Pereira Carneiro 80, Belo Horizonte 30510-010, MG, Brazil
2
Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Avenida Antônio Carlos 6627, Belo Horizonte 30123-970, MG, Brazil
*
Author to whom correspondence should be addressed.
Foods 2020, 9(10), 1368; https://doi.org/10.3390/foods9101368
Submission received: 11 August 2020 / Revised: 22 September 2020 / Accepted: 23 September 2020 / Published: 26 September 2020
(This article belongs to the Special Issue Detection of Residual Pesticides in Foods)
Versions Notes

Abstract

:
This study aimed to optimize and validate a multi-residue method for identifying and quantifying pesticides in honey by using both gas and liquid chromatographic separation followed by mass spectrometric detection. The proposed method was validated to detect 168 compounds, 127 of them by LC-MS/MS (liquid chromatography tandem mass spectrometric detection) and 41 by GC-MS/MS (gas chromatography tandem mass spectrometric detection). The limit of detection (LOD) and limit of quantification (LOQ) values for the analytes determined by LC-MS/MS were 0.0001–0.0004 mg/kg and 0.0002–0.0008 mg/kg, respectively. For GC-MS/MS analyses, the LOD and LOQ values were 0.001–0.004 mg/kg and 0.002–0.008 mg/kg. In total, 33 samples of commercial honey produced by apiaries in six Brazilian states were analyzed with the validated method. Residual amounts of 15 analytes were detected in 31 samples (93.9%). The method described in the present study was able to detect an extensive and broad range of pesticides with very high sensitivity.

1. Introduction

Honey is consumed by humans worldwide because of its characteristic sweet flavor and as a medicinal food. It is produced by honeybees, mainly from nectar collected from flowers. However, honey may be contaminated with pesticides used on crops foraged by bees. Contamination may occur through direct contact of the bee body to the pesticide or by bee consumption of the contaminated nectar, pollen, and guttation fluid (an exudate eliminated through the tips or edges of leaves of some plants) [1,2,3]. Furthermore, some pesticides are used to treat beehives against diseases [4].
The consumption of residual pesticides in contaminated foods has been linked to several toxic effects in humans, such as carcinogenesis, immunological disorders, and neurological disturbances [5]. Maximum residue levels (MRLs) have been established for pesticides in honey to ensure consumers’ safety [6,7,8,9]. It is mandatory to avoid the commercialization of honey containing residual pesticides at levels above the MRLs. To determine residual pesticide levels, precise and sensitive analytical methods must be able to detect an extensive and broad range of compounds.
Several analytical methods have been developed for detecting single compounds to a few dozen pesticides in honey. In these methods, detection and quantification are performed using techniques such as liquid chromatography (LC) with diode array [10], ultraviolet [11,12], fluorescence [13], and electrochemical [11] detectors, gas chromatography (GC) with electron capture [14], flame ionization [15], nitrogen–phosphorus [16], flame photometric [17], thermionic-specific [18], and atomic emission [19] detectors, and excitation–emission matrix fluorescence data [20].
The performance of chromatographic analysis depends on adequate sample extraction and cleanup procedures. Matrix compounds are concentrated at the extraction procedure, whereas interfering substances are removed by the cleanup procedure [21]. An innovative technique developed for sample extraction and cleanup procedures is the QuECHERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method [22]. Compared to earlier procedures, this method reduces the volume of solvents, and offers practical performance. Modifications of the QuECHERS method have been used for the detection of pesticides in different matrices such as meat [23], fish [24], milk [25], and honey [3,26,27,28,29,30,31].
Simultaneous detection of the residual levels of several pesticides in honey is mandatory in several countries to inspect this food before commercialization. Multi-residue analysis of at least one hundred pesticides in honey has been achieved using LC and GC coupled to mass spectrometric (MS) or tandem mass spectrometric (MS/MS) detection [1,3,26,27,28,29,30,31,32].
This study aimed to develop and validate a multi-residue method for identifying and quantifying pesticides in honey by using both gas and liquid chromatographic separation followed by mass spectrometric detection.

2. Materials and Methods

2.1. Chemicals and Reagents

Acetonitrile, ethyl acetate (both high performance liquid chromatography [HPLC] grade), and formic acid (for analysis) were supplied by Merck (Darmstadt, Germany). Methanol (HPLC grade) was obtained from Honeywell (Charlotte, NC, USA). Ammonium formate (>99%) was purchased from Vetec (Rio de Janeiro, Brazil). A DisQuETM CEN sample preparation kit in pouch format (each pouch containing 4.0 g of anhydrous magnesium sulfate, 1.0 g of sodium chloride, 1.0 g of trisodium citrate dihydrate, and 0.5 g of disodium hydrogen citrate sesquihydrate; all > 99%) was supplied by Waters (Milford, CT, USA). An ExtraBond® QuEChERS Dispersive kit EN (each tube containing 900 mg of anhydrous magnesium sulfate and 150 mg of primary and secondary amine (PSA); both > 99%) was obtained from Scharlab (Barcelona, Spain). D-Sorbitol (≥98%) and gluconolactone (>99%) were purchased from Sigma–Aldrich (Darmstadt, Germany). Ultrapure water was generated with a Millipore Milli-Q system (Milford, CT, USA). All reference standards were of high purity grade (>98.0%) and were obtained from Dr. Ehrenstorfer (Augsburg, Germany) or AccuStandard (New Haven, CT, USA). Individual stock solutions were prepared at an approximate concentration of 1000 ng/µL in acetonitrile or acetone and stored in a freezer at −20 °C. Working solutions were prepared through appropriate dilutions of the stock solutions.

2.2. Samples

Blank samples of honey were obtained from apiaries managed under an organic system, and repeated analyses confirmed the absence of residual pesticides. These blank samples were fortified with target analytes for the validation of the analytical method. Furthermore, 33 samples of commercial honey produced by apiaries in six Brazilian states (Distrito Federal, Goias, Minas Gerais, Rio Grande do Norte, Rio Grande do Sul, and São Paulo) were analyzed using the validated method.

2.3. Sample Preparation

The modified QuEChERS method for extraction and cleanup was optimized from previously described procedures [22,28,32,33]. Each honey sample (5.0 g) was placed into a 50 mL polypropylene tube and spiked with appropriate amounts of pesticides in working solutions. Next, 10.0 mL of ultrapure water was added, and the mixture was agitated at 1750 rpm for 2 min. Exactly 10.0 mL of a solution of acetonitrile and ethyl acetate (70:30, v/v) was added, and each tube was agitated again at 1750 rpm for 2 min. Then, 4.0 g of anhydrous magnesium sulfate, 1.0 g of sodium chloride, 1 g of trisodium citrate dehydrate, and 0.5 g of disodium hydrogen citrate sesquihydrate were added, and the tubes were agitated at 1750 rpm for another 2 min and centrifuged at 4000 rpm for 5 min. The whole organic layer was transferred to a 15 mL polypropylene tube, and the mixture was kept at −40 °C for at least 2 h. The supernatant (6.0 mL) was mixed with 900 mg of anhydrous magnesium sulfate and 150 mg of PSA, and the mixture was agitated at 1750 rpm for 1 min and centrifuged at 3600 rpm for 5 min. The extract (4.0 mL) was transferred to two 13 × 100 mm glass tubes, with 2.0 mL in each tube. The solution was dried in an evaporator with a water bath maintained at 45 °C and nitrogen pressure of 15 psi.
The procedural internal standard (P-IS) [34] for the LC analysis was Propoxur, and the P-IS for GC analysis was 4,4´-dichlorodiphenyldichloroethylene (4,4´-DDE). After weighing the honey sample, 10 μL of the P-IS solution containing 4.0 ng/μL of Propoxur and 4.0 ng/μL of DDE 4,4 was added. Propoxur and 4,4´-DDE were then validated following the validation method described in Section 2.3.
For LC analysis, the dried residue was reconstituted with 200 µL of methanol:water (1:1), with both solvents containing 5 mM ammonium formate and 0.01% formic acid. After 30 min, the tube was vortexed for 1 min, and the solution was transferred to a vial containing a conical insert of 250 μL.
For GC analysis, the dried residue was reconstituted with 200 µL of acetonitrile:ethyl acetate (7:3) and 6 µL of analyte protectant solution, composed of 10 mg/mL gluconolactone and 5 mg/mL D-sorbitol in acetonitrile:water (7:3). The tube was then immediately vortexed for 0.5 min, and the solution was transferred to a vial containing a conical insert of 250 μL.

2.4. Liquid Chromatography

LC-MS/MS (liquid chromatography tandem mass spectrometric detection) analysis was performed using an Agilent 6495 Triple Quadrupole LC/MS system. Chromatographic separations were carried out on a Zorbax SB-C18 Rapid Resolution HT column (4.6 × 150 mm, 1.8 µm) at a 40 °C column temperature. The mobile phases were water containing 5 mM ammonium formate and 0.01% formic acid (phase A) and methanol containing 5 mM ammonium formate and 0.01% formic acid (phase B), with gradient elution at a flow rate of 0.6 mL/min. The gradient elution program was as follows: 0 min, 90% B; 2.0 min, 50% B; 20 min, 100% B. The total chromatographic run time was 25 min. The injection volume was 5 µL.
For mass spectrometric analysis, an electrospray ionization (ESI) source was used in both negative (ESI-) and positive (ESI+) modes. Source parameters were set as follows: gas temperature 120 °C, gas flow 15 L/min, nebulizer 45 psi, sheath gas flow 12 L/min, sheath gas temperature 300 °C, capillary voltage 3500 V (+ and −), nozzle voltage 300 V (+)/500 V (−), iFunnel RF high pressure 150 V (+)/90 V (−), and iFunnel RF low pressure 60 V (+ and −). The retention times, delta retention times, polarities, ion transitions, and collision energies are presented in Table 1. Two transitions were chosen for almost all pesticides, but an extra confirmatory transition was included for four pesticides to avoid false-positives at trace pesticide levels. The analysis was run according to all requirements for identifying analytes by MS/MS established by European Union SANTE/12682/2019 [34].

2.5. Gas Chromatography

GC-MS/MS (gas chromatography tandem mass spectrometric detection) analysis was performed using an Agilent 7000C Triple Quadrupole GC/MS system with a multimode inlet. The temperature of the injector was maintained at 150 °C (0.1 min), ramped up to 300 °C at 600 °C/min (20 min hold), and then ramped down to 200 °C at 20 °C/min until the end of the analysis. The injection volume was 2 μL. The pulsed splitless injection was at 50 psi for 0.5 min with a split flow of 50 mL/min for 0.6 min. The gas saver was set to 20 L/min and started after 5 min. The carrier gas was helium, and the inlet pressure was 5.59 psi (constant pressure mode) during the run and 2.0 psi during the backflush. From the inlet, two Agilent HP-5ms Ultra Inert (5%-phenyl)-methylpolysiloxane columns (0.25 mm, 0.25 µm) were coupled to each other through a purged ultimate union for post-run backflushing; the first column was 30 m, and the second column was 2 m. The total chromatographic run time was 29.5 min, and backflushing started after 25.5 min with 8.92 psi. The column oven temperature was maintained at 60 °C for 1.0 min, ramped up to 180 °C at 30 °C/min, and then ramped up to 300 °C at 5 °C/min.
For the mass spectrometric analysis, an electron ionization source was used with an ionization voltage of 70 eV, ion source temperature of 290 °C, and interface temperature of 280 °C. The retention times, delta retention times, polarities, ion transitions, and collision energies are presented in Table 2. Two transitions were chosen for almost all pesticides, but an extra confirmatory transition was included for seven pesticides to avoid false-positives at trace pesticide levels. The analysis was run according to all requirements for identifying analytes by MS/MS established by European Union SANTE/12682/2019 [34].

2.6. Method Validation

Validation was performed following the European Union SANTE/12682/2019 [34] and Codex Alimentarius CXG90-2017 [35] guidelines. The following analytical performance parameters were assessed: linearity, selectivity, trueness, precision (repeatability and within-lab reproducibility), limit of detection (LOD), and limit of quantification (LOQ). A total of 209 different analytes were tested, 159 of them by LC-MS/MS and 50 by GC-MS/MS.
Matrix-matched calibration (MMC) was used to minimize the matrix effect. For the preparation of analytical MMC curves, blank honey extracts were spiked with appropriate amounts of standard solutions at the six final concentrations. Three independent solutions were prepared for each level of the curve (n = 18), and the samples were injected randomly. The difference between the calculated concentration and the theoretical concentration must be less than or equal to 20% for the curve’s best fit. The selectivity was determined by identifying the pesticide in the presence of the matrix and other analytes. If interfering peaks were detected at the same retention time as some pesticides, the interfering agents’ areas had to be less than or equal to 30% of the analyte LOQs.
The trueness and precision (repeatability and within-lab reproducibility) were determined from the recovery assay results of blank samples spiked with all of the analytes at two distinct levels (LOQ and 10× LOQ) for GC-MS/MS and three distinct levels (LOQ, 2× LOQ, and 10× LOQ) for LC-MS/MS. Repeatability was evaluated using data from replicate samples (n = 6) analyzed on the same day for each level. The within-lab reproducibility was evaluated using replicate data (n = 12) from two different days and two analysts for each level. Repeatability and within-lab reproducibility are expressed by the relative standard deviation (RSD in %), whereas average recovery values express trueness. The expanded measurement uncertainty (U) was estimated by the top-down approach. All results are reported in Table 3 and Table 4. Average recovery ranging from 70% to 120% was considered adequate. Precision deviations of up to 20% were considered acceptable [34].
The LOQ was determined as the lowest concentration level of the calibration curve with acceptable accuracy. The LOD corresponded to 50% of the estimated value for the quantification limit, provided that the recoveries presented an area greater than or equal to 50% of the point in the matrix solution injected and that the signal/noise ratio was higher than or equal to 3.

3. Results and Discussion

3.1. Extraction Method

The extraction procedure is a crucial step for detecting pesticides, and it can be challenging for a complicated matrix such as honey. Extraction procedures that have been developed for honey samples include solvent extraction, supercritical fluid extraction, solid-phase extraction, matrix solid-phase dispersion, solid-phase microextraction, stir bar sorptive extraction [36], purge and trap, dispersive liquid–liquid microextraction, microextraction by packed sorbent, single-drop microextraction, magnetic solid-phase extraction [37], and solvent floatation [38]. In the present method, the QuEChERS method was optimized for the extraction and cleanup of honey samples from the original method [22] with modifications for honey [28,33] and bee pollen samples [32]. The original QuEChERS method consists of an extraction step with acetonitrile and separation using extraction salts, followed by a cleanup step with purification salts [22].
Different extraction and cleanup conditions were evaluated for this method. Honey samples were diluted in water prior to extraction. Acetonitrile:ethyl acetate (70:30, v/v) solution provided better extraction efficiency, similar to Souza Tette et al. [28]. On the other hand, Mitchell et al. [33] used acetonitrile:water (50:50, v/v) solution without the sample’s previous dilution. In the present study, the extracted solution was subjected to freeze-out before the dispersive solid phase extraction (d-SPE) cleanup, following the method developed for bee pollen by Vázquez et al. [32]. The extraction recoveries for most pesticides were improved by keeping the extract in the freezer at −40 °C for at least 2 h (Supplementary Materials Table S1). Furthermore, extracted solutions that were subjected to freeze-out were visually more translucid than solutions that were not subjected to freeze-out.
The cleanup procedure of the present study was performed with magnesium sulfate and PSA. The same purification salts were also used by Mitchell et al. [33], but at different amounts (150 mg magnesium sulfate and 100 mg PSA); in contrast, Souza Tette et al. [28] also included Florisil (50 mg) to magnesium sulfate (150 mg) and PSA (50 mg). The extract was concentrated ten times after cleanup to achieve lower LOD and LOQ values, similarly to an earlier study [33]. The effectiveness of the modifications to the QuEChERS method in the present study was confirmed by the wide range of pesticides successfully detected and the high sensitivity evidenced by the low LOD and LOQ values.

3.2. Validation Assay

The proposed method was validated to detect 168 compounds, 127 of them by LC-MS/MS and 41 by GC-MS/MS. The matrix effect was minimized by using MMC. The method’s selectivity was determined by identifying the pesticide in the presence of the matrix and other analytes. All validated compounds showed average recoveries ranging from 70% to 120%. The mean repeatability relative standard deviation (RSD) for all samples in the LC-MS/MS method was 7.75%, ranging from 2% to 20%, and in the GC-MS/MS method the RSD was 7.24%, ranging from 3% to 15%. The expanded measurement uncertainty (U) for all samples in the LC-MS/MS method was 11.4%, ranging from 3% to 20%, and in the GC-MS/MS method was 13.1%, ranging from 4% to 20%. Average recoveries ranging from 70% to 120% and precision RSD of up to 20% were considered adequate [34]. The estimation of the uncertainty of an analytical method can be performed in different ways, including empirical, practical, or top-down approaches [39]. In the present study, the uncertainty was estimated using the top-down approach. In this way, the experimental design to estimate the RSD under conditions of partial reproducibility varied the day and the analysts to reproduce the variations.
Table 3 and Table 4 show the linearity, recovery, RSD, expanded measurement uncertainty (U), LOD, and LOQ results for analytes determined using LC-MS/MS and GC-MS/MS, respectively. The LOD and LOQ values for 119 analytes determined by LC-MS/MS were 0.0001 mg/kg and 0.0002 mg/kg, respectively, whereas seven analytes showed LOD and LOQ values of 0.0002 mg/kg and 0.0004 mg/kg, and the values for one analyte were 0.0004 mg/kg and 0.0008 mg/kg. For GC-MS/MS analyses, the LOD and LOQ values were 0.001 mg/kg and 0.002 mg/kg for nine analytes, 0.002 mg/kg and 0.004 mg/kg for 30 analytes, and 0.004.0 mg/kg and 0.008 mg/kg for two analytes.
A total of 41 analytes could not be validated, 32 of which were analyzed by LC-MS/MS and 9 by GC-MS/MS (Supplementary Materials Tables S2 and S3). These compounds were detected, but the obtained values for linearity, recovery rate, RSD, and U were not following the European Union SANTE/12682/2019 [34] and Codex Alimentarius CXG90-2017 [35] guidelines.
Pacífico da Silva et al. [1] developed an analytical method with an LC-MS/MS system for the simultaneous detection of 152 pesticides in honey after extraction with ethyl acetate and cleanup using Florisil. The LOD and LOQ values for all the tested pesticides were 0.005 and 0.01 mg/kg, respectively [1]. Paoloni et al. [40] used Florisil for sample cleanup after extraction with n-Hexane for determining 13 pesticides in honey using GC-MS/MS. The LOQ for all tested pesticides was 0.01 mg/kg, and the LOD was not provided [40]. Česnik et al. [31] used a GC-MS method for detecting 75 pesticides and an LC-MS/MS method for detecting 60 pesticides in honey after extraction with a mixture of petroleum ether and dichloromethane. The LOQ ranged from 0.01 to 0.05 mg/kg with the GC-MS method and from 0.003 to 0.01 mg/kg with the LC-MS/MS method [31].
The QuEChERS method was applied for pesticide extraction in honey by other authors [26,27,28,29,30,41,42]. The LC-MS/MS method described by Souza Tette et al. [28] was validated to measure 116 pesticides in honey, but 11 compounds showed recoveries at 0.010 mg/kg out of the 70–120% range. The LOD was 0.005 mg/kg and the LOQ varied between 0.01 and 0.025 mg/kg [28]. The LC-ESI-MS/MS method of Kasiotis et al. [26] detected 115 pesticides, but some analytes showed recoveries below 70%. The LOD ranged from 0.00003 to 0.0233 mg/kg, and the LOQ ranged from 0.0001 to 0.078 mg/kg [26]. Another LC-MS/MS method for analyzing honey samples was described for 207 pesticides [30], with LOQ values ranging from 0.001 to 0.01 mg/kg. However, the LOD was not reported, and some pesticides showed recoveries out of the 70–120% range [30]. In another LC-MS/MS method [29], 132 tested compounds were measured in honey, obtaining recoveries ranging from 70% to 120% for 116 compounds. However, the LOD and LOQ were not provided in the manuscript nor supplementary material [29]. The GC-MS/MS method described by Zheng et al. [41] was validated to measure six pesticides in honey. The LOD ranged from 0.0004 to 0.002 mg/kg and the LOQ varied between 0.001 and 0.005 mg/kg [41]. Another GC-MS/MS method was developed by Shendy et al. [27] for the detection of 200 pesticides in honey. The LOD ranged from 0.001 to 0.003 mg/kg and the LOQ was 0.005 to 0.01 mg/kg, but the recoveries ranged from 51.13–126.55% [27]. Both LC-MS/MS and GC-MS/MS analysis of residual pesticides in honey was described by Bargańska et al. [42]. This method was validated for 51 compounds, 18 of them determined by LC-MS/MS, 21 compounds by GC-MS/MS, and 12 compounds by both methods. The LOD ranged from 0.0028 to 0.09 mg/kg with the LC method and from 0.0023 to 0.027 mg/kg with the GC method [42]. Compared with these above articles, the method described in the present study was able to detect extensive and broad-spectrum pesticides (168) with very high sensitivity.

3.3. Real Samples

Of the 33 honey samples analyzed, 31 (93.9%) showed residual levels of pesticides (Table 5). Each sample contained up to 15 detected analytes. The most frequently detected compounds were carbendazim (20 samples), thiabendazole (20 samples), azoxystrobin (15 samples), chlorpyrifos (12 samples), and imidacloprid (12 samples). Carbendazim is a fungicide that is widely used in agriculture. Its toxic effects include liver damage, disruption of endocrine and hematological functions, and reproductive toxicity [43]. Thiabendazole is a fungicide and anthelmintic compound with hepatotoxic and teratogenic effects, and it is probably a carcinogen [44]. Azoxystrobin is also a fungicide, and its toxicity includes lesions in the liver and kidneys [45]. Chlorpyrifos is an organophosphate pesticide that is used as an insecticide and acaricide. It is considered moderately toxic and can cause disruption of neuronal, reproductive, immune, and endocrine systems, cancer, and chromosome damage [46]. Imidacloprid is a neonicotinoid insecticide that is highly toxic to honeybees [1,2], with neurotoxic, immunotoxic, teratogenic, and mutagenic effects in mammals [47]. The presence of pesticides in a considerable percentage of the analyzed samples is indicative of widespread environmental contamination by these compounds. However, the consumption of the analyzed honey may not be considered unsafe because the residual levels of all detected pesticides were below the MRLs established for Brazil [9] and the European Union [6,7,8].
Few studies have been aimed at determining the presence of residual pesticides in honey in Brazil. Organophosphorus trichlorfon was detected in just one sample from one hundred commercial honey samples from five states of Brazil [28]. A total of 19 pesticides were found in 53 honey samples collected directly from colonies in the Rio Grande do Norte state, northeastern Brazil. Thirteen of these pesticides were detected in honey produced by honeybees pollinating melon crops (23 samples); however, only six were found in honey from honeybees foraging in the forest (20 samples), and four in honey produced by the stingless bee Melipona subnitida (10 samples) [1]. In another study, honey produced by M. subnitida from the Rio Grande do Norte state was tested for residual pesticides. Of the 35 analyzed samples, 25 showed residual pesticides, and the detected compounds were chlorpyrifos-methyl, monocrotophos, and trichlorfon [3]. These data support the requirement for testing honey for the presence of pesticides to avoid commercialization of batches containing residual levels above the MRLs.

4. Conclusions

The proposed method was successfully optimized and validated for multi-residue identification and quantification of pesticides in honey. It was able to detect an extensive and broad range of pesticides with remarkably high sensitivity and precision. The developed method was successfully applied to Brazilian commercial honey, showing the analyzed honey was considered safe for consumption.

Supplementary Materials

The following are available online at https://www.mdpi.com/2304-8158/9/10/1368/s1, Table S1. Modified QuEChERS method optimization. Extraction with acetonitrile, or a solution of acetonitrile and ethyl acetate (70:30, v/v), and inclusion of a freezing out step prior to clean up (900 mg of anhydrous magnesium sulfate and 150 mg of PSA). Results are presented as recovery (in %) for each analyte of the LC-MS/MS; Table S2. Non-approved analytes. Linearity, recovery (in %), repeatability relative standard deviation (RSD; in %), expanded measurement uncertainty (U; in %), limit of detection (LOD; in mg/kg), and limit of quantification (LOQ; in mg/kg) for each analyte of the LC-MS/MS method for analysis of pesticides in honey; Table S3: Non-approved analytes. Linearity, recovery (in %), repeatability relative standard deviation (RSD; in %), expanded measurement uncertainty (U; in %), limit of detection (LOD; in mg/kg), and limit of quantification (LOQ; in mg/kg) for each analyte of the GC-MS/MS method for analysis of pesticides in honey.

Author Contributions

Conceptualization, M.O.A., S.C.S.O. and B.S.-B.; investigation, M.O.A., S.C.S.O., V.H.F.F., M.C.M.R. and D.M.C.; writing—original draft preparation, review and editing, B.S.-B.; funding acquisition, S.C.S.O. and B.S.-B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Fundação de Amparo à Pesquisa do Estado de Minas Gerais—FAPEMIG, grant numbers APQ-02304-16 and BIP-00056-17, and Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq, grant number 305761/2013-7.

Conflicts of Interest

The authors declare no conflict of interest.

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Table
Table 1. Chromatographic parameters and MS/MS (tandem mass spectrometric) detection for compounds analyzed by LC-MS/MS (liquid chromatography tandem mass spectrometric detection).
Table 1. Chromatographic parameters and MS/MS (tandem mass spectrometric) detection for compounds analyzed by LC-MS/MS (liquid chromatography tandem mass spectrometric detection).
NameRT 1 (min)DRT 2 (min)PolarityTransitionsCollision Energy
2,4-D9.601.5ESI−QI 3 218.9 > 161.11
1st CI 4 218.9 > 124.935
2nd CI 220.9 > 162.911
Acephate4.561.5ESI+QI 184.0 > 143.05
1st CI 184.0 > 125.015
Acetamiprid6.561.0ESI+QI 223.1 > 126.015
1st CI 223.1 > 56.015
Aldicarb8.051.5ESI+QI 208.1 > 116.210
1st CI 208.1 > 89.124
Aldicarb-Sulfone4.991.5ESI+QI 223.1 > 148.05
1st CI 223.1 > 76.05
Aldicarb-Sulfoxide4.881.5ESI+QI 207.1 > 131.90
1st CI 207.1 > 89.18
Allethrin19.361.5ESI+QI 303.2 > 135.010
1st CI 303.2 > 12320
Ametryn13.151.5ESI+QI 228.1 > 186.120
1st CI 228.1 > 116.128
2nd CI 228.1 > 96.025
Aminocarb7.321.5ESI+QI 209.1 > 152.212
1st CI 209.1 > 137.220
Atrazine11.341.5ESI+QI 216.1 > 174.116
1st CI 216.1 > 68.040
Avermectin B1a21.711.5ESI+QI 890.5 > 567.48
1st CI 890.5 > 305.116
Azaconazole11.031.5ESI+QI 300.0 > 231.116
1st CI 300.0 > 159.028
Azinphos-Ethyl14.741.5ESI+QI 346.1 > 132.112
1st CI 346.1 > 97.032
Azinphos-Methyl12.151.5ESI+QI 318.0 > 261.00
1st CI 318.0 > 132.18
Azoxystrobin12.901.5ESI+QI 404.1 > 372.112
1st CI 404.1 > 344.128
2nd CI 404.1 > 329.136
Benalaxyl16.861.5ESI+QI 326.2 > 294.14
1st CI 326.2 > 148.127
Bitertanol16.981.5ESI+QI 338.2 > 99.110
1st CI 338.2 > 70.04
Boscalid13.341.5ESI+QI 343.0 > 307.116
1st CI 343.0 > 271.232
Bromacil9.291.5ESI+QI 261.0 > 205.020
1st CI 261.0 > 187.940
Bromuconazole15.503.0ESI+QI 378.0 > 159.032
1st CI 378.0 > 70.035
Buprofezin19.131.5ESI+QI 306.2 > 201.15
1st CI 306.2 > 116.110
Cadusafos17.961.5ESI+QI 271.1 > 130.920
1st CI 271.1 > 97.040
Carbaryl9.801.5ESI+QI 202.1 > 145.14
1st CI 202.1 > 127.128
Carbendazim7.071.5ESI+QI 192.1 > 160.116
1st CI 192.1 > 132.132
Carbofuran9.351.5ESI+QI 222.1 > 165.120
1st CI 222.1 > 123.130
3-Hydroxycarbofuran6.351.5ESI+QI 238.1 > 220.10
1st CI 238.1 > 163.18
Carboxin9.881.5ESI+QI 236.1 > 143.112
1st CI 236.1 > 93.136
Chlorfenvinphos17.201.5ESI+QI 358.9 > 155.08
1st CI 358.9 > 99.228
Chlorfluazuron20.001.5ESI+QI 539.9 > 383.044
1st CI 539.9 > 158.036
Chlorpyrifos20.201.5ESI+QI 349.9 > 198.020
1st CI 349.9 > 97.020
Chlorpyrifos-Methyl-Oxon15.891.5ESI+QI 334.0 > 306.08
1st CI 334.0 > 278.08
Clofentezine16.991.5ESI+QI 303.0 > 138.012
1st CI 303.0 > 102.040
Clomazone12.701.5ESI+QI 242.1 > 127.020
1st CI 240.1 > 125.020
2nd CI 240.1 > 89.156
Clothianidin6.081.5ESI+QI 250.0 > 169.08
1st CI 250.0 > 131.98
Cyanazine8.431.5ESI+QI 241.1 > 214.118
1st CI 241.1 > 104.044
Cyanofenphos16.601.0ESI+QI 304.1 > 276.012
1st CI 304.1 > 157.024
Cyazofamid15.431.5ESI+QI 325.0 > 261.04
1st CI 325.0 > 108.08
Cymoxanil6.971.5ESI+QI 199.1 > 128.04
1st CI 199.1 > 110.912
Cyproconazole14.802.0ESI+QI 292.1 > 125.032
1st CI 292.1 > 70.016
Cyprodinil17.101.5ESI+QI 226.1 > 108.030
1st CI 226.1 > 93.040
Cyromazine4.481.5ESI+QI 167.1 > 125.016
1st CI 167.1 > 85.016
Diafenthiuron20.821.5ESI+QI 385.2 > 329.216
1st CI 385.2 > 278.232
Diazinon17.101.5ESI+QI 305.1 > 169.132
1st CI 305.1 > 97.040
Dichlorvos9.111.5ESI+QI 221.0 > 109.012
1st CI 221.0 > 79.024
Dicrotophos5.831.5ESI+QI 238.0 > 127.012
1st CI 238.0 > 112.18
Difenoconazole17.801.5ESI+QI 406.1 > 337.018
1st CI 406.1 > 251.028
Diflubenzuron14.961.5ESI+QI 311.0 > 158.08
1st CI 311.0 > 141.032
Dimethoate6.531.5ESI+QI 230.0 > 198.80
1st CI 230.0 > 125.016
Dimethomorph13.803.0ESI+QI 388.1 > 301.120
1st CI 388.1 > 165.132
Diniconazole18.001.5ESI+QI 326.1 > 159.028
1st CI 326.1 > 70.028
Disulfoton17.691.5ESI+QI 275.0 > 89.012
1st CI 275.0 > 61.044
Disulfoton-Sulfone10.721.5ESI+QI 307.0 > 125.010
1st CI 307.0 > 97.030
Disulfoton-Sulfoxide10.781.5ESI+QI 291.0 > 185.010
1st CI 291.0 > 157.020
Diuron11.381.5ESI+QI 2350. > 72.020
1st CI 233.0 > 160.024
2nd CI 233.03 > 72.120
Emamectin B1a20.892.0ESI+QI 886.5 > 158.044
1st CI 886.5 > 82.164
Emamectin B1b20.342.0ESI+QI 872.5 > 158.340
1st CI 872.5 > 82.368
Epoxiconazole15.211.5ESI+QI 330.1 > 121.016
1st CI 330.1 > 101.252
Ethion19.651.5ESI+QI 385.0 > 199.14
1st CI 385.0 > 142.824
Etofenprox22.821.5ESI+QI 394.2 > 359.05
1st CI 394.2 > 177.05
Ethoprophos15.721.5ESI+QI 243.1 > 130.915
1st CI 243.1 > 97.030
Etrimfos16.801.5ESI+QI 293.1 > 265.026
1st CI 293.1 > 125.028
Famoxadone16.931.5ESI+QI 392.1 > 330.94
1st CI 392.1 > 238.012
Fenamiphos15.921.5ESI+QI 304.1 > 234.012
1st CI 304.1 > 217.120
Fenbuconazole15.111.5ESI+QI 337.1 > 125.140
1st CI 337.1 > 70.033
Fenpyroximate20.911.5ESI+QI 422.2 > 366.212
1st CI 422.2 > 135.036
Fenthion16.861.5ESI+QI 279.0 > 247.18
1st CI 279.0 > 169.112
Fipronil15.501.5ESI+QI 437.0 > 368.018
1st CI 437.0 > 255.026
Flazasulfuron11.241.5ESI+QI 408.1 > 182.128
1st CI 408.1 > 83.040
Fluazifop-Butyl18.801.5ESI+QI 384.1 > 328.112
1st CI 384.1 > 282.220
Flufenoxuron19.881.5ESI+QI 489.1 > 158.020
1st CI 489.1 > 140.956
Fluquinconazole15.111.5ESI+QI 376.0 > 349.020
1st CI 376.0 > 307.124
Flutriafol11.171.5ESI+QI 302.1 > 122.928
1st CI 302.1 > 70.116
Furathiocarb19.301.5ESI+QI 383.2 > 251.98
1st CI 383.2 > 195.016
Heptenophos11.791.5ESI+QI 251.0 > 127.015
1st CI 251.0 > 125.025
Hexaconazole17.401.5ESI+QI 314.1 > 159.030
1st CI 314.1 > 70.120
Hexythiazox19.901.5ESI+QI 353.1 > 227.98
1st CI 353.1 > 168.124
Imazalil14.303.0ESI+QI 297.1 > 201.015
1st CI 297.1 > 159.020
Imazapyr5.483.0ESI+QI 262.1 > 217.120
1st CI 262.1 > 131.040
Imazethapyr7.421.5ESI+QI 290.1 > 245.124
1st CI 290.1 > 177.029
Imibenconazole19.601.5ESI+QI 411.0 > 171.020
1st CI 411.0 > 125.040
Imidacloprid5.971.5ESI+QI 258.0 > 210.912
1st CI 256.0 > 208.912
2nd CI 256 > 17512
Indoxacarb17.881.5ESI+QI 528.1 > 203.045
1st CI 528.1 > 150.020
Iprodione15.981.5ESI+QI 330.0 > 287.910
1st CI 330.0 > 244.914
Iprovalicarb15.201.5ESI+QI 321.2 > 202.95
1st CI 321.2 > 11916
Kresoxim-Methyl16.501.5ESI+QI 314.1 > 267.00
1st CI 314.1 > 222.110
Linuron12.671.5ESI+QI 249.0 > 160.120
1st CI 249.0 > 133.036
Lufenuron19.031.5ESI+QI 510.9 > 158.020
1st CI 510.9 > 141.057
Malaoxon9.371.5ESI+QI 315.0 > 127.120
1st CI 315.0 > 99.24
Malathion14.301.5ESI+QI 331.0 > 126.95
1st CI 331.0 > 99.010
Metalaxyl11.931.5ESI+QI 280.2 > 220.110
1st CI 280.2 > 160.120
Metconazole17.301.5ESI+QI 320.1 > 125.048
1st CI 320.1 > 70.124
Methamidophos4.311.5ESI+QI 142.0 > 125.010
1st CI 142.0 > 94.010
Methidathion11.951.5ESI+QI 302.9 > 145.00
1st CI 302.9 > 85.115
Methiocarb13.141.5ESI+QI 226.1 > 169.04
1st CI 226.1 > 121.112
Methomyl5.441.5ESI+QI 163.1 > 106.04
1st CI 163.1 > 88.00
Methoxyfenozide14.081.5ESI+QI 369.2 > 313.10
1st CI 369.2 > 149.010
Metolachlor16.301.5ESI+QI 284.1 > 252.18
1st CI 284.1 > 176.124
Metribuzin9.381.5ESI+QI 215.1 > 187.115
1st CI 215.1 > 84.030
Mevinphos7.353.0ESI+QI 225.0 > 193.10
1st CI 225.0 > 127.012
Monocrotophos5.521.5ESI+QI 224.1 > 193.00
1st CI 224.1 > 127.010
Myclobutanil14.001.5ESI+QI 289.1 > 125.132
1st CI 289.1 > 70.116
Naled11.801.5ESI+QI 380.7 > 127.08
1st CI 380.7 > 109.024
Omethoate4.771.5ESI+QI 214.0 > 125.016
1st CI 214.0 > 109.024
Oxamyl5.091.5ESI+QI 237.1 > 90.010
1st CI 237.1 > 72.012
Paclobutrazol13.951.2ESI+QI 294.1 > 125.240
1st CI 294.1 > 70.120
Paraoxon10.881.5ESI+QI 276.1 > 220.010
1st CI 276.1 > 94.040
Paraoxon-Methyl8.051.5ESI+QI 248.0 > 201.920
1st CI 248.0 > 90.025
Parathion16.401.5ESI+QI 292.0 > 236.18
1st CI 292.0 > 94.140
Penconazole16.701.5ESI+QI 284.1 > 15930
1st CI 284.1 > 70.115
Pencycuron18.001.5ESI+QI 329.1 > 125.124
1st CI 329.1 > 89.160
Pendimethalin20.201.5ESI+QI 282.1 > 212.14
1st CI 282.1 > 194.116
Phenthoate16.201.5ESI+QI 321.0 > 163.18
1st CI 321.0 > 79.144
Phorate17.501.5ESI+QI 261.0 > 199.02
1st CI 261.0 > 75.15
Phosmet12.801.5ESI+QI 317.9 > 160.08
1st CI 317.9 > 133.036
Phosphamidon8.301.5ESI+QI 300.0 > 174.18
1st CI 300.0 > 127.116
Picloram4.711.5ESI+QI 243.0 > 196.822
1st CI 243.0 > 169.834
2nd CI 241 > 222.810
Picoxystrobin16.031.5ESI+QI 368.1 > 205.24
1st CI 368.1 > 145.020
Pirimicarb11.081.5ESI+QI 239.1 > 182.112
1st CI 239.1 > 72.120
Pirimiphos-Ethyl19.391.5ESI+QI 334.1 > 198.122
1st CI 334.1 > 182.124
Pirimiphos-Methyl18.001.5ESI+QI 306.2 > 164.120
1st CI 306.2 > 108.130
Prochloraz18.031.5ESI+QI 376.0 > 308.04
1st CI 376.0 > 265.912
Profenofos18.831.5ESI+QI 374.9 > 347.012
1st CI 374.9 > 304.919
Propamocarb4.721.5ESI+QI 189.2 > 144.08
1st CI 189.2 > 102.012
Propargite20.261.5ESI+QI 368.1 > 231.20
1st CI 368.1 > 175.28
Propiconazole17.501.5ESI+QI 342.1 > 159.032
1st CI 342.1 > 69.116
Propoxur9.331.5ESI+QI 210.11 > 168.15
1st CI 210.11 > 111.18
Pyraclostrobin17.501.5ESI+QI 388.11 > 193.88
1st CI 388.11 > 163.120
Pyrazophos17.261.5ESI+QI 374.1 > 222.116
1st CI 374.1 > 194.132
Pyridaben21.901.5ESI+QI 365.1 > 309.14
1st CI 365.1 > 147.220
Pyridaphenthion14.361.5ESI+QI 341.0 > 205.110
1st CI 341.0 > 189.020
Pyrimethanil13.881.5ESI+QI 200.1 > 106.920
1st CI 200.1 > 82.025
Pyriproxyfen19.901.5ESI+QI 322.2 > 227.212
1st CI 322.2 > 185.020
2nd CI 322.2 > 9612
Quinalphos16.481.5ESI+QI 299.0 > 163.020
1st CI 299.0 > 147.020
Quizalofop-Ethyl19.101.5ESI+QI 373.0 > 271.224
1st CI 373.0 > 255.136
Simazine9.451.5ESI+QI 202.1 > 132.022
1st CI 202.1 > 124.126
Spinosyn A19.872.0ESI+QI 732.5 > 142.128
1st CI 732.5 > 98.160
Spinosyn D20.782.0ESI+QI 746.5 > 142.135
1st CI 746.5 > 98.055
Spirodiclofen21.251.5ESI+QI 411.1 > 3138
1st CI 411.1 > 71.215
Spiromesifen20.671.5ESI+QI 371.2 > 273.112
1st CI 371.2 > 255.124
Sulfentrazone9.381.5ESI+QI 404.0 > 306.928
1st CI 404.0 > 273.040
Tebuconazole16.801.5ESI+QI 308.1 > 124.947
1st CI 308.1 > 70.040
Tebufenozide16.081.5ESI+QI 353.2 > 297.14
1st CI 353.2 > 133.020
Teflubenzuron19.001.5ESI+QI 381.0 > 158.012
1st CI 381.0 > 141.048
Temephos18.901.5ESI+QI 467.0 > 419.020
1st CI 467 > 124.944
Terbufos19.601.5ESI+QI 289.1 > 233.00
1st CI 289.1 > 57.116
Tetraconazole15.091.5ESI+QI 372.0 > 159.036
1st CI 372.0 > 70.020
Thiabendazole8.221.5ESI+QI 202.0 > 175.024
1st CI 202.0 > 131.036
Thiacloprid7.101.5ESI+QI 253.0 > 126.016
1st CI 253.0 > 90.040
Thiamethoxam5.421.5ESI+QI 292.0 > 211.18
1st CI 292.0 > 181.120
Thiobencarb18.031.5ESI+QI 258.0 > 125.125
1st CI 258.07 > 100.15
Thiodicarb10.591.5ESI+QI 355.0 > 108.18
1st CI 355.0 > 88.18
Thiophanate-Methyl8.651.5ESI+QI 343.0 > 151.020
1st CI 343.0 > 93.056
Tolylfluanid15.991.5ESI+QI 346.9 > 238.112
1st CI 346.9 > 137.025
Triadimefon14.801.5ESI+QI 294.1 > 197.28
1st CI 294.1 > 69.116
Triadimenol14.701.5ESI+QI 296.1 > 99.116
1st CI 296.1 > 70.012
Triazophos14.301.5ESI+QI 314.1 > 162.116
1st CI 314.1 > 119.136
Trichlorfon6.551.5ESI+QI 256.9 > 221.04
1st CI 256.9 > 109.012
Trifloxystrobin18.351.5ESI+QI 409.1 > 186.012
1st CI 409.1 > 145.052
Triflumizole18.501.5ESI+QI 346.1 > 278.04
1st CI 346.1 > 43.120
Vamidothion6.391.5ESI+QI 288.0 > 146.16
1st CI 288.0 > 58.044
Zoxamide16.961.5ESI+QI 336.0 > 187.016
1st CI 336.0 > 159.044
1 RT: retention time. 2 DRT: delta retention time. 3 QI: quantification ions. 4 CI: confirmation ions.
Table
Table 2. Chromatographic parameters and MS/MS detection for compounds analyzed by GC-MS/MS (gas chromatography tandem mass spectrometric detection).
Table 2. Chromatographic parameters and MS/MS detection for compounds analyzed by GC-MS/MS (gas chromatography tandem mass spectrometric detection).
NameRT 1 (min)DRT 2 (min)Quantification TransitionCollision Energy
Alachlor11.331QI 3 188.1 > 160.110
1st CI 4 188.1 > 130.140
Aldrin12.621QI 263.0 > 193.030
1st CI 298.0 > 263.08
Bifenthrin19.782QI 182.0 > 167.012
1st CI 181.0 > 165.025
Bromophos-Methyl13.061QI 330.9 > 315.916
1st CI 329.0 > 314.016
Bromopropylate19.822QI 341.0 > 185.05
1st CI 341.0 > 183.020
Carbophenothion17.711QI 153.0 > 96.910
1st CI 153.0 > 79.030
2nd CI 157.0 > 75.140
Cyfluthrin 24.392QI 162.9 > 127.05
1st CI 226.9 > 77.130
Cypermethrin25.012QI 162.9 > 127.05
1st CI 181.1 > 127.135
Clordane Gama (Trans)14.422QI 272.0 > 237.016
1st CI 375.0 > 266.025
Chlorfenapyr161QI 247.0 > 227.015
1st CI 247.0 > 200.025
2nd CI 247.0 > 197.05
Chlorothalonil10.171QI 265.9 > 230.920
1st CI 263.8 > 229.020
2nd CI 263.8 > 168.025
Chlorpyrifos-Methyl11.142QI 288.0 > 93.026
1st CI 288.0 > 273.015
2nd CI 286.0 > 271.016
Chlorthiophos16.931QI 297.0 > 269.014
1st CI 269.0 > 205.016
2,4´-DDD15.71QI 237.0 > 165.020
1st CI 235.0 > 165.020
2,4´-DDE14.472QI 246.0 > 176.030
1st CI 248.0 > 211.020
4,4´-DDE15.921QI 246.0 > 176.030
1st CI 248.0 > 176.020
2,4´-DDT16.841QI 237.0 > 165.020
1st CI 235.0 > 165.020
4,4´-DDT181QI 237.0 > 165.020
1st CI 235.0 > 165.020
Deltamethrin27.941QI 253.0 > 93.020
1st CI 253.0 > 174.015
Dicofol18.462QI 253.0 > 141.015
1st CI 249.9 > 139.110
Dieldrin15.691QI 263.0 > 191.035
1st CI 263.0 > 193.035
Endosulfan Alpha14.842QI 238.8 > 204.015
1st CI 241.0 > 206.015
Endosulfan Beta16.351QI 241.0 > 206.015
1st CI 195.0 > 159.015
Endosulfan Sulfate17.941QI 271.9 > 236.915
1st CI 240.8 > 205.915
Endrin16.061QI 263.0 > 191.035
1st CI 263.0 > 193.035
Esfenvalerate26.912QI 225.0 > 119.015
1st CI 167.0 > 125.010
Fenpropathrin20.11QI 265.0 > 210.015
1st CI 265.0 > 89.035
2nd CI 181.0 > 152.026
Fenarimol21.941QI 139.0 > 111.015
1st CI 219.0 > 107.010
Fenitrothion11.941QI 277.0 > 260.05
1st CI 277.1 > 109.020
2nd CI 276.8 > 125.015
Phosalone20.911QI 182.0 > 111.015
1st CI 182.0 > 75.140
HCH Alpha9.12QI 180.9 > 145.012
1st CI 218.8 > 183.05
HCH Beta9.572QI 180.9 > 145.012
1st CI 218.8 > 183.05
HCH Delta10.381QI 180.9 > 145.012
1st CI 218.8 > 183.05
HCH Gamma9.812QI 180.9 > 145.012
1st CI 218.8 > 183.05
Heptachlor11.631QI 271.9 > 236.825
1st CI 274.0 > 239.020
Heptacloro Exo Epoxid13.711QI 353.0 > 263.015
1st CI 353.0 > 282.015
Hexachlorobenzene (HCB)9.231QI 283.9 > 213.935
1st CI 283.9 > 248.825
Lambda Cyhalothrin21.651QI 181.1 > 152.130
1st CI 197.0 > 161.010
Methoxychlor202QI 227.0 > 141.140
1st CI 227.0 > 169.020
Mirex21.681QI 271.9 > 235.025
1st CI 272.0 > 237.020
Ovex (Clorfenson)15.111QI 174.8 > 111.110
1st CI 177.0 > 113.012
2nd CI 302.0 > 175.04
Oxyfluorfen15.641QI 252.0 > 146.032
1st CI 252.0 > 170.032
Parathion-Methyl11.282QI 263.0 > 109.115
1st CI 263.0 > 79.130
Permethrin23.382QI 183.1 > 153.115
1st CI 183.0 > 115.225
Procymidone13.972QI 283.0 > 96.010
1st CI 283.0 > 67.140
Prothiofos15.211QI 162.0 > 63.140
1st CI 267.0 > 239.05
Quintozene9.731QI 249.0 > 214.020
1st CI 295.0 > 237.020
Tetradifon20.711QI 226.9 > 199.010
1st CI 355.7 > 159.010
Trifluralin8.481QI 306.0 > 264.010
1st CI 263.9 > 160.115
Vinclozolin11.222QI 212.0 > 172.015
1st CI 212.0 > 109.040
1 RT: retention time. 2 DRT: delta retention time. 3 QI: quantification ions. 4 CI: confirmation ions.
Table
Table 3. Linearity, recovery (in %), repeatability relative standard deviation (RSD; in %), expanded measurement uncertainty (U; in %), limit of detection (LOD; in mg/kg), and limit of quantification (LOQ; in mg/kg) for each analyte of the LC-MS/MS method for analysis of pesticides in honey.
Table 3. Linearity, recovery (in %), repeatability relative standard deviation (RSD; in %), expanded measurement uncertainty (U; in %), limit of detection (LOD; in mg/kg), and limit of quantification (LOQ; in mg/kg) for each analyte of the LC-MS/MS method for analysis of pesticides in honey.
CompoundLinearityAverage RecoveryRSDULODLOQ
Type of AdjustPonderationLR 1 (µg/kg)Pt 2 1Pt 2Pt 6Pt 1Pt 2Pt 6Pt 1Pt 2Pt 6(mg/kg)(mg/kg)
3-HydroxycarbofuranLinear 1–10110115926686680.000100.00020
AcephateLinear1/x1–1010991814105810100.000100.00020
AcetamipridLinear1/x1–10101961011074201980.000100.00020
AldicarbLinear1/x1–10959886811121215120.000100.00020
Aldicarb-sulfoneLinear 1–1011810490511651160.000100.00020
Aldicarb-sulfoxideLinear 1–101181111007119814170.000100.00020
AllethrinLinear 1–109280831018201018200.000100.00020
AmetrynLinear 1–10119106993446490.000100.00020
AminocarbLinear1/x1–1097100901394149120.000100.00020
AtrazineLinear 1–10107104103454141060.000100.00020
AzaconazoleLinear 1–10108107108121041211100.000100.00020
Azinphos-ethylLinear 1–10104106968778890.000100.00020
Azinphos-methylLinear 1–101199888934914140.000100.00020
AzoxystrobinLinear1/x1–1010593997341420160.000100.00020
BenalaxylLinear 1–101191099767810780.000100.00020
BitertanolLinear 1–10111103944857850.000100.00020
BoscalidLinear 1–10119978810461220140.000100.00020
BromacilLinear1/x1–1010095926641619120.000100.00020
BromuconazoleLinear 2–201181081014547540.000200.00040
BuprofezinLinear1/x1–10102103881817141917140.000100.00020
CadusafosLinear 1–1011010292571197110.000100.00020
CarbarylLinear 1–10116959510741012110.000100.00020
CarbendazimLinear1/x1–10114108114683712120.000100.00020
CarbofuranLinear1/x1–1010699101423918150.000100.00020
ChlorfenvinphosLinear1/x1–1011111010846510650.000100.00020
ChlorpyrifosLinear 1–109179741418161618160.000100.00020
Chlorpyrifos-methyl-oxonLinear 1–101131081004696690.000100.00020
ClofentezineLinear 1–101171059269991290.000100.00020
ClomazoneLinear1/x1–1010588998551219150.000100.00020
ClothianidinLinear 1–1011910510056610860.000100.00020
CyanazineLinear 1–101189590947919190.000100.00020
CyanofenphosLinear1/x1–1010510296915111015110.000100.00020
CyazofamidLinear 1–101059994745191890.000100.00020
CyproconazoleLinear1/x²2–2010511011534215740.000200.00040
CyprodinilLinear 1–1010010695718111318110.000200.00040
DicrotophosLinear1/x1–1010492935541319160.000100.00020
DifenoconazoleLinear 1–101131051005785890.000100.00020
DiflubenzuronLinear 1–10113106101391059100.000100.00020
DimethoateLinear 1–1010010099884101240.000100.00020
DimethomorphLinear 1–101191091025356850.000100.00020
DiniconazoleLinear1/x1–10971019778101012110.000100.00020
Disulfoton-sulfoneLinear 1–1011010910634510860.000100.00020
DiuronLinear1/x1–101121041104103131140.000100.00020
Emamectin B1aLinear 1–101141181066758850.000100.00020
Emamectin B1bLinear 1–10113112111896131060.000100.00020
EpoxiconazoleLinear 1–101091101028558680.000100.00020
EthionLinear1/x²1–10848887919201619200.000100.00020
EthoprophosLinear 1–10107101926786790.000100.00020
EtrimfosLinear1/x1–10106100977578690.000100.00020
FamoxadoneLinear1/x1–101069888611121611120.000100.00020
FenbuconazoleLinear1/x1–1098100108565201780.000100.00020
FenpyroximateLinear 1–1011310395694613200.000100.00020
FenthionLinear1/x1–10106949051081912110.000100.00020
FipronilLinear1/x²2–2010610199511861180.000200.00040
Fluazifop-P-butylLinear1/x1–10106877781216912160.000100.00020
FluquinconazoleLinear 1–1010610611076610860.000100.00020
FurathiocarbLinear 1–101079688461066100.000100.00020
HeptenophosLinear 1–10104979477889120.000100.00020
HexaconazoleLinear1/x1–10105108100696141190.000100.00020
HexythiazoxLinear 1–10978777516152016150.000100.00020
ImazalilLinear1/x1–10106102103946171680.000100.00020
ImibenconazoleLinear1/x1–1089.585.879.94.89.913.620.012.913.60.000100.00020
ImidaclopridLinear 1–10999192101041018180.000100.00020
IndoxacarbLinear 1–10105958751111611110.000100.00020
IprodioneLinear1/x²1–1094101951410151411160.000100.00020
IprovalicarbLinear 1–10103106105474161470.000100.00020
Kresoxim-methylLinear 1–10112104966646980.000100.00020
LinuronLinear 1–101179190181131819100.000100.00020
MalaoxonLinear 1–1011811710019107191180.000100.00020
MalathionLinear 1–101021041094342019160.000100.00020
MetalaxylLinear1/x1–101081051072441414110.000100.00020
MetconazoleLinear 1–10111106944795790.000100.00020
MethidathionLinear1/x1–10108100969451012110.000100.00020
MethiocarbLinear 1–10120100917631020190.000100.00020
MethomylLinear1/x1–10928588101061920200.000100.00020
MethoxyfenozideLinear 1–10110108993676670.000100.00020
MetolachlorLinear1/x1–10105105100549201290.000100.00020
MetribuzinLinear1/x1–1096979815952010120.000100.00020
MonocrotophosLinear 1–101089082131561516170.000100.00020
MyclobutanilLinear 1–1011010810353511550.000100.00020
OmethoateLinear 1–1095847875381060.000100.00020
OxamylLinear 1–1099101100101031917100.000100.00020
PaclobutrazolLinear 1–101079897633109100.000100.00020
ParaoxonLinear 1–1011111911710851015150.000100.00020
ParathionLinear1/x4–409499931611141611140.000400.00080
PenconazoleLinear 1–1010711310386410860.000100.00020
PencycuronLinear1/x1–10928689712151212150.000100.00020
PendimethalinLinear 1–109882801215131415130.000100.00020
PhenthoateLinear 1–101091019351012710150.000100.00020
PhosmetLinear 1–101071041085441416100.000100.00020
PhosphamidonLinear 1–101181081004347650.000100.00020
PicoxystrobinLinear 1–1011010711065715970.000100.00020
PirimicarbLinear1/x1–101151101113249440.000100.00020
Pirimiphos-ethylLinear 1-1010290899813168130.000100.00020
Pirimiphos-methylLinear1/x1–1010410195710111014130.000100.00020
ProchlorazLinear1/x1–101081061063761210110.000100.00020
ProfenofosLinear1/x²2–201039895510101010120.000200.00040
PropargiteLinear 1–1010488731218191218190.000100.00020
PropoxurLinear1/x1–10108989510451520190.000100.00020
PyraclostrobinLinear 1–101109996710111012130.000100.00020
PyrazophosLinear 1–10111104964776770.000100.00020
PyridaphenthionLinear 1–101171111025646640.000100.00020
PyriproxyfenQuadratic1/x²1–109892931815172015170.000100.00020
QuinalphosLinear 1–101101059857867100.000100.00020
Quizalofop-P-ethylLinear 1–1096888281015810170.000100.00020
SimazineLinear1/x1–10104103965651518140.000100.00020
Spinosyn ALinear 1–101051061014565560.000100.00020
Spinosyn DLinear 1–10106101971317141717160.000100.00020
SpirodiclofenLinear 1–109984841119161119200.000100.00020
SpiromesifenLinear 1–108681731012171012170.000100.00020
TebuconazoleLinear 1–109610210074812980.000100.00020
TebufenozideLinear 1–10106100841110161110160.000100.00020
TeflubenzuronLinear1/x2–201049081913181013180.000200.00040
TerbufosLinear1/x²2–208674711312111913110.000200.00040
TetraconazoleLinear1/x1–10102105105566911100.000100.00020
ThiabendazoleLinear1/x1–101111149517185191850.000100.00020
ThiaclopridLinear 1–10117103986536630.000100.00020
ThiamethoxamLinear1/x1–1010694918768980.000100.00020
ThiobencarbLinear1/x1–101069491117813990.000100.00020
ThiodicarbLinear 1–1011198971034101490.000100.00020
TolylfluanidLinear 1–10100959685712880.000100.00020
TriadimefonLinear1/x1–101031061046851413110.000100.00020
TriadimenolLinear 1–1011611610643569110.000100.00020
TriazophosLinear1/x1–101131041094531413110.000100.00020
TrichlorfonLinear1/x²1–10107101979759990.000100.00020
TrifloxystrobinLinear 1–10105969351112911120.000100.00020
TriflumizoleLinear 1–109394929713107130.000100.00020
VamidothionLinear1/x1–101059290674121560.000100.00020
ZoxamideLinear 1–101049288698910100.000100.00020
1 LR: linearity range. 2 Pt: point.
Table
Table 4. Linearity, recovery (in %), repeatability relative standard deviation (RSD; in %), expanded measurement uncertainty (U; in %), limit of detection (LOD; in mg/kg), and limit of quantification (LO Q; in mg/kg) for each analyte of the GC-MS/MS method for analysis of pesticides in honey.
Table 4. Linearity, recovery (in %), repeatability relative standard deviation (RSD; in %), expanded measurement uncertainty (U; in %), limit of detection (LOD; in mg/kg), and limit of quantification (LO Q; in mg/kg) for each analyte of the GC-MS/MS method for analysis of pesticides in honey.
CompoundLinearityAverage RecoveryRSDULODLOQ
Type of AdjustPonderationFT (µg/kg)Pt 1Pt 6Pt 1Pt 6Pt 1Pt 6(mg/kg)(mg/kg)
DDE 4,4Linear1/x10–10011994872070.0010.002
AlachlorLinear 10–1001031097519120.0010.002
AldrinLinear1/x20–2001109910918110.0020.004
AzoxystrobinLinear 10–1009878971380.0010.002
BifenthrinLinear 20–20011791341140.0020.004
Bromophos-methylLinear 20–2001191006715160.0020.004
BromopropylateLinear1/x20–20011391661470.0020.004
CarbophenothionLinear1/x20–20011593551750.0020.004
CyfluthrinLinear1/x40–40010692581380.0040.008
CypermethrinLinear 20–20010289781680.0020.004
Clordane gama (trans)Linear1/x20–200112997518100.0020.004
ChlorfenapyrLinear1/x20–200103101852060.0020.004
ChlorfenvinphosLinear 10–1001209810416130.0010.002
Chlorpyrifos-methylLinear1/x10–10011310115920180.0010.002
ChlorthiophosLinear1/x20–20011894961860.0020.004
DDD 2,4Linear1/x10–100115951071470.0010.002
DDT 2,4Linear1/x10–10011298572080.0010.002
DDT 4,4Linear1/x20–20010998551970.0020.004
DeltamethrinLinear1/x²10–1009611913420100.0010.002
DieldrinLinear1/x20–200113961072080.0020.004
DifenoconazoleLinear1/x10–1009985971680.0010.002
Endosulfan alphaLinear1/x20–20011698851780.0020.004
Endosulfan betaLinear1/x20–20011195561870.0020.004
Endosulfan sulfateLinear1/x20–200108103751860.0020.004
EndrinLinear1/x20–200111981051960.0020.004
EsfenvalerateLinear1/x20–200100105861990.0020.004
FenpropathrinLinear1/x20–20010992551650.0020.004
FenarimolLinear1/x20–200105856712100.0020.004
FipronilLinear1/x20–20010910411419150.0020.004
FluquinconazoleLinear1/x10–10010890751360.0010.002
PhosaloneLinear1/x20–20010292871870.0020.004
HCH alphaLinear1/x20–2001078481113110.0020.004
HeptachlorLinear1/x20–20010890121118160.0020.004
Hexachlorobenzene (HCB)Linear1/x10–100988012913130.0010.002
IprodioneLinear1/x20–200108891381580.0020.004
Lambda cyhalothrinLinear1/x20–200110101761660.0020.004
MethoxychlorLinear1/x20–20010897561960.0020.004
MirexLinear1/x10–1001131085918180.0010.002
ChlorfensonLinear1/x20–20010993961780.0020.004
OxyfluorfenLinear1/x²20–2001081135619150.0020.004
PendimethalinLinear1/x10–100991046618180.0010.002
PermethrinLinear1/x20–20099861151860.0020.004
PirimicarbLinear1/x10–10011510413614160.0010.002
Pirimiphos-ethylLinear1/x10–1001021057518160.0010.002
ProcymidoneLinear1/x20–200103966710100.0020.004
ProfenofosLinear1/x20–2001121024514130.0020.004
ProthiofosLinear1/x20–20011397851580.0020.004
QuintozeneLinear1/x10–1001068791418140.0010.002
TetradifonLinear1/x40–40010790471580.0040.008
TrifluralinLinear1/x20–2001129491115140.0020.004
VinclozolinLinear1/x20–200111101678140.0020.004
Table
Table 5. Detected pesticides (in mg/kg) in 33 samples of honey using the developed LC-MS/MS and GC-MS/MS method.
Table 5. Detected pesticides (in mg/kg) in 33 samples of honey using the developed LC-MS/MS and GC-MS/MS method.
CompoundPositive SamplesMaximum LevelsLOD 1LOQ 2MRL 3
Acephate80.007790.00010.00020.020
Acetamiprid1<LQ0.00010.00020.050
Azoxystrobin150.000190.00010.00020.050
Bifenthrin3<LQ0.0020.0040.010
Boscalid1<LQ0.00010.00020.050
Carbaryl20.000500.00010.00020.050
Carbendazim200.003500.00010.00021.0
Clomazone5<LQ0.00010.0002-
Chlorpyrifos120.000340.00010.00020.010
Clothianidin20.000630.00010.0002-
Diflubenzuron30.000260.00010.00020.050
Dimethoate60.001940.00010.00020.010
Diuron5<LQ0.00010.00020.050
Imidacloprid120.006180.00010.00020.050
Metoxyphenazide1<LQ0.00010.00020.050
Omethoate2<LQ0.00010.00020.010
Pyraclostrobin2<LQ0.00010.00020.050
Pyrimethanil30.000400.00010.0002-
Pyriproxyfen3<LQ0.00010.00020.050
Tebuconazole100.000450.00010.00020.050
Thiabendazole200.001300.00010.00020.010
Thiamethoxam90.002090.00010.00020.050
Triazophos1<LQ0.00010.00020.010
Trifloxystrobin50.000300.00010.00020.050
1 LOD: limit of detection (in mg/kg). 2 LOQ: limit of quantification (in mg/kg). 3 MRL: maximum residue level (in mg/kg) [9].

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MDPI and ACS Style

Almeida, M.O.; Oloris, S.C.S.; Faria, V.H.F.; Ribeiro, M.C.M.; Cantini, D.M.; Soto-Blanco, B. Optimization of Method for Pesticide Detection in Honey by Using Liquid and Gas Chromatography Coupled with Mass Spectrometric Detection. Foods 2020, 9, 1368. https://doi.org/10.3390/foods9101368

AMA Style

Almeida MO, Oloris SCS, Faria VHF, Ribeiro MCM, Cantini DM, Soto-Blanco B. Optimization of Method for Pesticide Detection in Honey by Using Liquid and Gas Chromatography Coupled with Mass Spectrometric Detection. Foods. 2020; 9(10):1368. https://doi.org/10.3390/foods9101368

Chicago/Turabian Style

Almeida, Mariana O., Silvia Catarina S. Oloris, Vanessa Heloisa F. Faria, Márcia Cassimira M. Ribeiro, Daniel M. Cantini, and Benito Soto-Blanco. 2020. "Optimization of Method for Pesticide Detection in Honey by Using Liquid and Gas Chromatography Coupled with Mass Spectrometric Detection" Foods 9, no. 10: 1368. https://doi.org/10.3390/foods9101368

APA Style

Almeida, M. O., Oloris, S. C. S., Faria, V. H. F., Ribeiro, M. C. M., Cantini, D. M., & Soto-Blanco, B. (2020). Optimization of Method for Pesticide Detection in Honey by Using Liquid and Gas Chromatography Coupled with Mass Spectrometric Detection. Foods, 9(10), 1368. https://doi.org/10.3390/foods9101368

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