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A comparison between QuEChERS and other pesticide extraction procedures for honey and honey bee matrices is discussed. Honey bee matrix was extracted by solvent based procedure whereas solid phase extraction was the protocol for the honey matrix. The citrate buffered QuEChERS method was used for both matrices. The methods were evaluated regarding cost (equipment and reagents), time, accuracy, precision, sensitivity and versatility. The results proved that the QuEChERS protocol was the most efficient method for the extraction of the selected pesticides in both matrices.
Inject 5 μL of the extract in the LC–MS/MS according to the conditions already reported  and detailed below.
Ionization and fragmentation settings were optimized by direct injection of pesticide standard solutions. MS/MS was performed in the SRM mode using ESI in positive mode. For each compound, two characteristic product ions of the protonated molecule [M+H]+ were monitored, the first and most abundant one was used for quantification, while the second one was used as a qualifier. Collision energy and cone voltage were optimized for each pesticide (Table 1). Nitrogen was used as collision, nebulising and desolvation gas. The ESI conditions were: capillary voltage 4000 V, nebulizer 15 psi, source temperature 300 °C and gas flow 10 L min−1. In order to maximize sensitivity, dynamic MRM was used, with MS1 and MS2 at unit resolution and cell acceleration voltage of 7 eV for all the compounds.
In order to compare QuEChERS to other routine procedures, methods were validated according to the European Union Guideliness . Furthermore, the main elements of uncertainty as the amount of sample used for a determination, the recovery value of the analytical procedure and the repeatability of determinations for a true sample , were considered through the validation process (for detailed information of the validation parameters, see Supplementary material Table S1 and S2).
The sensitivity of the method was estimated by establishing the limits of quantification (LOQs) (Fig. 1). The LOQs were determined in pure solvent and in spiked honey and honey bees samples. LOQs were calculated as the lowest concentration or mass of the analyte that has been validated with acceptable accuracy by applying the complete analytical method. LOQs were from 0.2 to 10 ng g−1 and from 0.03 to 10 ng g−1 for honey and honey bee matrices respectively. Solvent and SPE methods were slightly more sensitive than QuEChERS approach.
Matrix effects were evaluated by comparing the slope of the previous calibration curve and the slope of that prepared in the extract of honey or honey bee matrix with six concentration levels of standard solutions (Fig. 2). Matrix effects were mostly suppressive in both matrices and ranged from −60 to 50 and from −60 to 35% in honey and honey bee matrices, respectively.
Mean recovery (as accuracy) and relative standard deviation (as precision) were evaluated by spiking the samples at the LOQ and 10 x LOQ, with a minimum of 5 replicates (Fig. 3). Recovery values of honey bee matrix were from 34 to 96%, whereas RSDs were in all cases <20%. Honey matrix showed recoveries that ranged from 30 to 96% and RDS were <20% except for 17 compounds that were from 21 to 42%. QuEChERS approach showed better results than solvent method in the honey bee matrix while SPE was slightly better both in accuracy and precision than QuEChERS extraction procedure for honey.
The use of pesticides in agricultural cropping systems is often discussed as a factor influencing honey bee health . Furthermore, honey, which is considered a healthy natural product, can be contaminated during its production from both agricultural and beekeeping practices , . The development of extraction procedures able to process samples in an economic way is crucial.
This paper presents some of the currently applied sample preparation methods for the separation and pre-concentration of pesticides in honey and honey bee samples. The composition of honey and honey bees is very different but both are complex matrices. In order to achieve an accurate and reliable analytical result, an efficient pre-concentration/separation step is usually required prior to determination, even when such a sensitive detection method as LC–MS/MS is used.
From an analytical point of view, honey can be considered as a highly concentrated sugar solution (mostly fructose). Then, after water dilution it can be extracted using protocols similar to those applied to water as SPE. The protocol described here requires a medium cost in reagent and equipment because the SPE sorbents involve a high cost. The extraction of a sample requires between 60 and 90 min, being evaporation the step that takes more time. The performance of the method provides the best sensitivity and lower matrix effects.
On the contrary, honey bees are rich in lipids and proteins, requiring most sophisticated and extensive sample preparation methods. Traditional methods as the solvent approach are long, tedious and require high amounts of expensive organic solvents . Considering the use of reagents and equipment this method has high cost, requires between 150 and 180 min to process a sample and provides recoveries slightly lower for more polar pesticides
The results pointed out that QuEChERS approach is used in many different matrices as hive products (beeswax, pollen, honey, honey bee) , , . Honey and honey bee composition (Fig. 4) evidence the versatility of the QuEChERS method compared to other extraction procedures as those used in the present work. Appropriate results in terms of specificity, selectivity, accuracy and sensitivity, low cost and quickness make QuEChERS a suitable procedure for determining pesticides in less studied hive matrices as royal jelly and propolis. Furthermore, QuEChERS approach meets important components of green analytical chemistry  due to its small amounts of solvent needed compared to the traditional methods.
This work has been supported by the agreement No. OTR2013-11072ASESO between the Agrupación de Defensa Sanitaria Apicola (apiADS) and the Environmental and Food Safety Research Group (SAMA-UV), Department of Medicine Preventive, Faculty of Pharmacy, University of Valencia (Spain).
MethodsX thanks the reviewers of this article for taking the time to provide valuable feedback.
Appendix ASupplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mex.2016.05.005.
The following are Supplementary data to this article: