Standard methods for Apis mellifera honey research

References (277)

1. Organochlorines (a-, b-, and Ç-hexachlorocyclohexane (HCH), hexachlorobenzene (HCB), aldrin, p,p'-DDE, p,p'- DDD, o,p'-DDT and p,p'-DDT)
2. Add 20 mL of ethyl acetate to the sample. Repeat the liquid- liquid extraction two times with 15 mL of ethyl acetate.
3. Centrifuge at 3000 rpm for 10 min if emulsion is formed.
4. Filtrate the organic phase through anhydrous sodium sulfate.
5. Evaporate the organic phase under a stream of nitrogen to 2.5 mL for analysis in graduated centrifuge tube.
6. Analyze by GC-ECD, and Gas Chromatography/Mass Spectrometry (GC-MS) for confirmatory analysis following authors' protocols.
7. Blasco, Lino et al. (2004)
8. Coumaphos, bromopropylate, amitraz and tau-fluvalinate
9. Mix 20 g of honey in an Ultra-Turrax blender with a mixture of n-hexane (60 mL), propanol-2 (30 mL) and 0.28% of ammonia. The pH of this mixture is 8.
10. Filter the solution through a filter paper.
11. Repeat steps 1 and 2 with the mixture of n-hexane (60 mL), propanol-2 (30 mL) and 0.28% of ammonia.
12. Rinse the Ultra-Turrax with 40 mL of n-hexane and filter this washing solution on the same filter paper as in step 2.
13. Add 50 mL of distilled water and 0.28% of ammonia (pH 10).
14. Shake the separating funnel vigorously.
15. Allow the filtrate to separate into two phases.
16. Discard the aqueous phase (lower).
17. Analyze by HPLC-DAD following authors' protocol. Martel and Zeggane (2002) analyte under examination, such as GC-MS and LC-MS (De Brabander et al., 2009). Some compounds such as nitroimidazoles (Polzer et al., 2010), fumagillin (Daeseleire & Reybroeck, 2012;
18. Kanda et al., 2011; Tarbin et al., 2010), and nitrofuran residues (Khong et al., 2004; Lopez et al., 2007; Tribalat et al., 2006) are mostly directly screened in honey using LC-MS/MS detection, since no or only few immuno- chemical methods for the detection in honey have been developed. By the development of multiresidue meth- ods, LC-MS is used more and more for multiclass screening for antimicrobial residues in honey (Azzouz & Ballesteros, 2015; Galarini et al., 2015; Hammel et al., 2008; Lopez et al., 2008).
19. Table 19. Extraction and test protocol (laboratory test) for the Chloramphenicol ELISA (EuroProxima B.V.). Extraction protocol 1. Bring 10 ± 0.1 g of honey into a centrifugation tube of 50 mL; fill also a centrifugation tube with the same amount of blank honey, honey doped with 0.3 mg/kg of chloramphenicol, and honey doped with 0.1 mg/kg of chloramphenicol, respectively.
20. Add to each sample 5 mL of distilled water.
21. Vortex the samples until all honey is dissolved.
22. Add 5 mL of ethyl acetate.
23. Evaporate at 45 ± 5 C under a mild stream of nitrogen.
24. Dissolve the residue in 2 mL of n-hexane for defatting purposes and vortex.
25. Add 1 mL of reconstitution buffer and vortex for 20 s.
26. After centrifugation (10 min at 2600 g), pipette the layer underneath (reconstitution buffer, ± 800 mL) into a short glass test tube.
27. Add again 1 mL of n-hexane and vortex for 20 s.
28. Add 1 mL of reconstitution buffer and vortex for 20 s.
29. After centrifugation (10 min at 2,600 g), pipette the layer underneath (reconstitution buffer, ± 800 mL) into a short glass test tube.
30. Use the extract in the ELISA (extract could be stored for one day at 4 ± 2 C). Test protocol 1. Before starting the test, the reagents should be brought up to ambient temperature by taking them out of the refrigerator $ 20 min before use. Keep the substrate away from light. After analysis, store the remaining reagents as soon as possible in the refrigerator.
31. Identify the wells of the microtiterstrip upon the plate configuration sheet.
32. Pipette 100 mL of reconstitution/zero standard buffer into well A1 (blank).
33. Pipette 50 mL of reconstitution/zero standard buffer into well A2 (zero standard).
34. Pipette 50 mL of chloramphenicol-free extract buffer into wells B1 and B2 (blank control sample).
35. Pipette 50 mL of extract of samples doped at 0.1 mg/kg into wells C1 and C2 (positive control sample 0.1 ppb).
36. Pipette 50 mL of extract of samples doped at 0.3 mg/kg into wells C1 and C2 (positive control sample 0.3 ppb).
37. Pipette 50 mL of each sample extract in duplicate into the remaining wells of the microtiter plate.
38. Add 25 of conjugate (CAP-HRPO) into all wells except in well A1.
39. Add 25 of antibody solution into all wells except in well A1.
40. Cover the plate with aluminum foil and shake the plate for 1 min.
41. Discard the solution from the microtiter plate and wash three times with rinsing buffer. Fill all the wells each time with rinsing buffer to the rim. Place the inverted plate on absorbent paper and tap the plate firmly to remove residual washing solution. Take care that none of the wells dry out before the next reagent is dispensed.
42. Shake the substrate solution before use. Pipette 100 mL of substrate solution into each well. Cover the plate and shake the plate slowly.
43. Incubate for 30 min in the dark at room temperature (20-25 C).
44. Add 10 mL of stop solution into each well.
45. Read the absorbance values (OD) immediately at 450 nm in a spectrophotometer. Interpretation of results
46. Subtract the optical density (OD) value of the blank well (A1) from the individual OD of the other wells.
47. Calculate the Cut-off by adding 3xSD of repeatability for the positive control sample spiked at 0.3 ppb (value calculated out of the validation data) to the mean of both corrected OD values for the two positive control samples spiked at 0.3 ppb.
48. Interpret the control samples: the negative control sample should test negative and the positive control sample spiked at 0.1 ppb should give an OD below the OD of the negative control and higher than the OD of the positive control sample at 0.3 ppb. The run is invalid if the control samples are not giving correct results.
49. Compare the corrected value for each sample to the cut-off value. If the corrected OD of the sample is equal to or below the cut-off: the sample is considered as suspect for the presence of chloramphenicol at 0.3 mg/kg; if the corrected OD of the sample is higher than the cut-off: the sample is free from residues of chloramphenicol at 0.3 mg/kg.
50. Table 20. Extraction and test protocol for Charm II Macrolide Honey Test (Charm Sciences Inc.). Honey sample preparation 1. Label a 50 mL conical centrifuge tube for each sample.
51. Add 20 grams of honey to an appropriately labeled centrifuge tube. Also, prepare two negative control samples by using blank honey and one positive control sample of blank honey spiked with 20 mg/kg of erythromycin A.
52. Add 30 mL MSU Extraction Buffer to each tube. Mix well until honey is completely dissolved by putting the samples for 30 min on a shaker or by vortexing the tubes.
53. Add 9-10 drops of M2-buffer. Check pH with pH indicator strips; pH of extract should be equivalent to 7.5 (À8.0) on pH strip.
54. If the pH is still too low, add M2 Buffer dropwise, mix, and retest pH until the desired pH is reached. Note: If pH is high, add 0.3 mL (300 mL) 0.1 M HCl, mix, and retest. If pH is still high, add 0.1 M HCl drop-wise, mix, and retest.
55. Using the syringe assembly, filter the entire sample through a glass fiber filter (e.g., Millipore-Millex AP prefilter of 25 mm).
56. Collect the entire sample into a clean container (50 mL conical tube or beaker).
57. After the sample has been pushed through, detach the filter holder. Rinse syringe and bivalve with 10 mL deionized water. Clean-up over C18 cartridge
58. Activate the C18 cartridge by pushing through 5.0 mL of methanol. The cartridge should be used within 10 min of activation.
59. Repeat the washing step with 5.0 mL of water.
60. Perform the extraction by adding the filtered honey solution to the syringe. Push the solution slowly through the preactivated C18 cartridge one drop at a time. The sample may be thick and difficult to push through the cartridge. Hold cartridge with two hands to prevent cartridge from popping off due to backpressure. Discard liquid that flows through the cartridge.
61. Wash the cartridge with 5.0 mL distilled water and discard the flow through.
62. Remove the C18 cartridge from the assembly and add 3.0 mL methanol directly into the cartridge. Attach cartridge to the assembly and bring labeled test tube in position.
63. Slowly push methanol one drop at a time through the cartridge and collect the eluate in a labeled test tube.
64. Dry eluate for each sample. Dry under nitrogen or air in a 40-45 C heat block or water bath and remove from the heat block or water bath when methanol is completely evaporated.
65. Once the methanol is completely evaporated, reconstitute the dried eluate with 5 mL of Zero Control Standard (ZCS) and vortex extensively.
66. Label test tubes and scintillation vials. Let Charm II reagents reach room temperature.
67. Add the white tablet to the empty test tube.
68. Add 300 ± 100 lL water. Mix 10 s to break up the tablet. Take additional time if required to be sure the tablet is broken up.
69. Add 5 ± 0.25 mL diluted sample or control. Use a new tip for each sample. Immediately mix by swirling sample up and down 10 times for 10 s.
70. Incubate at 65 ± 2 C for 2 min.
71. Add green tablet (< 0.19kBq C 14 labeled erythromycin). Immediately mix by swirling the sample up and down 10 times for 15 s. The tablet addition and mixing of all samples should be completed within 40 s.
72. Incubate at 65 ± 2 C for 2 min.
73. Immediately pour off liquid completely. While draining, remove fat ring and wipe dry with swabs. Do not disturb the pellet.
74. Add 300 ± 100 lL water. Mix thoroughly to break up the pellet. The pellet must be suspended in water before adding scintillation fluid.
75. Add 3.0 ± 0.5 mL of scintillation fluid. Cap and invert (or shake) until mixture has a uniform cloudy appearance.
76. Count in liquid scintillation counter for 60 s. Read cpm (counts per minute) on [14C] channel. Count within 10 min of adding of scintillation fluid.
77. Calculate the Control Point by taking the highest negative control CPM À35%.
78. Recount if greater than and within 50 cpm of the Control Point. Interpretation of results
79. The cpm for the positive control sample should be below the Control Point, otherwise the run is not valid.
80. If the sample cpm is greater than the Control Point, the sample is negative. Report as "Not Found". If the sample cpm is less than or equal to the Control Point, the sample is positive. The presence of macrolides/lincosamides could be confirmed by a confirmatory method (LC-MS/MS).
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