الفهرس 
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Abstract
As beeswax and honey are widely used in nutrition, cosmetics and pharmaceuticals, they should contain minimal amounts of contaminants. Therefore, studying pesticide residues in beeswax and honey is relevant not only to beekeeping issues but also to economic, environment and to public health purposes.
Therefore, the aim of this study was to develop and validate an analytical method for determination of a high number of pesticide residues (373) in beeswax with direct injection into both GC–MS/MS and LC-MS/MS techniques. On the other hand, another method was developed and validated to determine (313) pesticide residues in honey using only LC-MS/MS. Finally, the validated methods were applied for the analysis of commercial beeswax and honey samples, collected from the Egyptian market.
The results obtained can be summarized as following:
5.1 Sample preparation and clean up
For beeswax, some organic solvents like (acetone, acetonitrile (ACN), ethyl acetate (EtAC), and mixtures of acetonitrile/ ethyl acetate at different ratios) were tested for melting the beeswax. It was observed that a mixture of ACN/ EtAC at the ratio of 1:3 can melt beeswax samples at a lower temperature of 60 ℃ for 20 min. Therefore, this mixture, of ACN/ EtAC at the ratio of 1:3, was selected in the current study as the best acceptable solvent extraction for pesticide residues analysis in beeswax.
The final extraction method procedure and clean up can be summarized as following: Two grams of the beeswax sample were placed in 50 mL polypropylene. Twenty mL of ACNEtAC (1:3, v/v) was added. Then after, these tubes were placed in a heated water bath at 60 °C for 20 min, shaking at 120 rpm. These tubes were centrifuged for 10 min at 4500 rpm, and then placed in the freezer at −20 °C for 2 h. After that, these samples were centrifuged again for 10 min at 4500 rpm. Two mL from the supernatant were collected and further cleaned in a 15 mL polypropylene tube containing PSA (0.20 g) and MgSO4 (1.00 g). These tubes were shaken for 1 min and centrifuged for 5 min at 4500 rpm. Finally, an aliquot was collected into the injection vials, using a 0.45 μm PTFE filter, to be ready for direct injection into LC-MS/MS and GC–MS/ MS.
For honey, the final extraction method procedure and clean up can be briefly described as following: Aliquots of 5.00 g were accurately weighed into 50 mL polypropylene centrifuge tubes. Ten ml of deionized water was added to the sample then incubated in a water bath at 40 oC, shaking at 120 rpm until complete homogeneity was obtained. Ten mL of (ACN: EtAC) (1:1, v/v) was added and the content was shaken for 1 min using a mechanical shaker. The QuEChERS salt kit (4.00 g anhydrous magnesium sulfate, 1.00 g sodium chloride, 1.00 g sodium citrate and 0.50 g sodium hydrogen citrate sesquihydrate) was added and immediately shaken for further 1 min and subsequently centrifuged for 5 min at 4500 rpm. Thereafter, the acetonitrile fraction (upper layer) was transferred to a 15 mL d- SPE polypropylene tube containing (1 g anhydrous MgSO4 and 0.2 g PSA) for cleanup. The tube was shaken for 1 min and centrifuged for 2 min at 4500 rpm. Finally, an aliquot was collected into vials, using a 0.45 μm PTFE filter, to be ready for direct injection into LC-MS/MS.
5.2. Methods validation.
Two different analytical methods were developed and validated for determination of 373 and 313 pesticide residues in beeswax and honey samples, respectively. LC-MS/MS and GC–MS/MS techniques were used in the validation of the analytical method for covering wide range of pesticide residues (373) in beeswax. These pesticides include 213 and 67 pesticides analyzed by LC-MS/MS and GC–MS/MS, respectively. In addition, there are 93 common pesticides were analyzed by LC-MS/MS and GC–MS/ MS for confirmation and quality control requirements. On the other hand, only LC-MS/MS technique was used in the development of an analytical method for determination of 313 pesticides in honey. The analytical methods validation of LC-MS/ MS and GC-MS/ MS for analysis of the studied pesticide residues in beeswax and honey was assessed by determining several analytical parameters according to SANTE/11813/2017 European Union guidelines.
Practical limits of quantification (LOQ) of pesticide residues in beeswax method were found to be varied between (20 – 100µg/kg), where there are 265 and 139 pesticides that can be determined at a very lower concentration of 20 µg/kg using LC-MS/ MS and GC-MS/ MS, respectively. On the other hand, the practical limits of quantification were found to be varied between (4 - 20 µg/kg) for honey method, where there are 270 (86%) of the studied pesticides in honey that can be determined at a lower concentration of 4 µg/kg using LC-MS/ MS. All these practical LOQs values were found to be lower than the maximum permissible limits of pesticides stated by Egyptian and Europeans standards.
The recovery tests were performed using five replicates of beeswax at three different concentrations levels (20, 50 and 100 µg/kg). Most of the studied pesticide residues in beeswax have Rec between 80 and 110% with good reproducibility (most of them have RSD below 8%). While For honey, the recovery tests were performed using five replicates at two different concentrations levels (4 and 20 µg/kg). Most of the studied pesticides in honey have Rec between 75 and 120% with good reproducibility (most of them have RSD below 12%).
For method linearity evaluations, five calibration levels 2, 10, 20, 50 and 100 µg/L were prepared in beeswax extract and honey extract for beeswax and honey analysis, respectively. Most of the studied pesticides in both beeswax and honey have correlation coefficients values greater than 0.995.
5.3. Pesticides determination in real samples of beeswax and honey.
A total of twenty-five samples of beeswax, and honey (ten beeswax, and fifteen honeys) collected from local Egyptian markets. All samples were subjected to pesticide residues analysis by the developed methods. Various pesticides concentrations were detected in analyzed beeswax samples. Malathion, chlorpyrifos, and cypermethrin were detected in all analyzed beeswax samples. Tau-fluvalinate, the commonly used insecticides/acaricides, was detected in 60% of the tested samples at high concentration levels ranged between <LOQ-1730 µg/kg. The obtained results from the analysis of real beeswax samples confirm the presence of an uncontrolled pesticide application to the beehive and in the surrounding agricultural area. On the other hand, all studied compounds were not detected in amounts above the LOQ for all analyzed honey samples except for two samples that was found contaminated by 2,4-dimethylphenylformamide (DMF) at concentration levels of 20 and 26 µg/kg. That result bears non associated heath risk to the consumer, since the maximum residual limit (MRL) of DMF in honey is 50µg/kg according to EU guidelines.