3.2.1. JH sample purification and quantification by GC-MSD

1. Clean all glassware by washing in acetone and hexane (3 times each) then baking overnight at 150°C (see section 3.1.1) prior to use.

2. Prepare solutions of:

   2.1. Ethyl ether: hexane 10:90 (v/v).
   2.2. Ethyl ether: hexane 30:70 (v/v).
   2.3. Ethyl acetate: hexane 50:50 (v/v).
   All solvents should be HPLC grade.
   2.4. Store at -20 °C until use.

3. Prepare a labelled 8 ml borosilicate glass vial with a Teflon lined cap for each sample.

4. Add 1.5 ml of 50 % aqueous acetonitrile to each vial.

5. Dilute 200 pg of farnesol (Sigma-Aldrich) in 10 µl hexane.

Farnesol will serve as an internal standard.

6. Add farnesol to the vials prepared at step 4.

7. After collecting a 2-4 µl sample of haemolymph (see section 3.1.1), expel it into the prepared sample vial.

8. Prepare a positive control vial, containing 200 pg JH III (Sigma-Aldrich) in 0.5 ml of 50% aqueous acetonitrile.

9. Prepare a negative control vial with just 50 % aqueous acetonitrile.

10. Add 2.5 ml hexane to each sample, using a graduated glass pipette.

11. Mix thoroughly with a vortexer.

12. The JH should partition into the upper hexane layer.

If the layers fail to separate well, centrifuge the vials at 1000 rpm for 1 min.

13. Using a flint glass Pasteur pipette, remove the hexane layer and transfer it into a fresh 8-ml vial.

14. Repeat the partitioning process twice, adding  2.5 ml hexane each time, for a total volume of 7.5 ml hexane extract.

15. Discard the bottom layer after the third extraction step.

16. Dry the hexane extracts completely by vacuum centrifugation or under a nitrogen stream (UHP grade).

17. While the samples are drying, prepare glass columns under a laboratory hood:

   17.1. Insert a small plug of glass wool at the narrow end of a Pasteur pipette that is sufficient to plug the column, but not so tightly packed as to impede the solvent flow rate.
   17.2. Place the pipettes in the holes of a column holder with a drip tray underneath.
   17.3. Add water [6 % (v/w)] to Al2O3 powder (activated, neutral, Brockmann I; Sigma-Aldrich).
   17.4. Mix until completely dry.
   17.5. Add 2 ml of the activated Al2O3 to the columns.
   17.6. Add 750 µl of hexane to the columns
   If the columns drip, add activated Al2O3 until they hold the volume.
   17.7. Wash the columns twice with 900 µl of hexane, allowing the hexane to drip into the tray.

18. Add 300 µl of hexane to each sample vial.

19. Cap.

20. Mix thoroughly to dissolve JH from the vessel walls.

21. Transfer each JH sample to a column, using clean glass pipettes.

22. Wash each sample vial twice more, adding 300 µl of hexane each time, thus transferring a total volume of 900 µl per sample to each column.

23. Add another 900 µl of hexane to each column to remove any remaining nonpolar compounds.

24. Wash each column twice with 900 µl of ethyl ether: hexane (10:90), allowing the flow-through to pass into the drip tray.

25. Wash each column with 750 µl of ethyl ether: hexane (30:70), again allowing the flow-through to drip into the tray.

26. Elute each column with 900 µl of ethyl ether: hexane (30:70) and collect the flow-through in new 8 ml vials.

27. Repeat step 26 once and pool both eluates in the same vial, for a total volume of 1.8 ml.

28. Discard the columns.

29. Concentrate the samples to dryness by vacuum centrifugation (~15 min) or under a nitrogen stream.

30. While drying the samples, equilibrate the ampoules containing the methanol-d4 to room temperature.

Methanol-d4 can absorb water from air, which may quench the reaction; raising the ampoules’ temperature reduces the likelihood of condensation forming.

31. With a micropipettor slowly add 75 µl of methanol-d4 to each sample vial.

32. Using a micropipettor, add 53 µl of trifluoroacetic acid (TFA) (spectrophotometric grade) to a 1 ml methanol-d4 ampoule.

33. Mix gently to make enough 5 % trifluoroacetic acid:methanol-d4 solution for 12 samples.

When doing so, evacuate air from the TFA container with nitrogen before storage.

34. Slowly add 75 µl of 5 % trifluoroacetic acid:methanol-d4 to each sample.

35. Tightly cap the vials and gently mix the contents.

36. Heat the sample vials for 20 min at 60°C to produce d3-methoxyhydrin derivatives.

37. Prepare new columns as described above (step 17).

38. Remove the samples from the oven (step 36).

39. Add 500 μl of hexane to each vial.

40. Mix well to remove residue from the walls.

41. Concentrate the samples to dryness by vacuum centrifugation (~15 min) or under a nitrogen stream.

42. Add 300 µl of hexane to each vial containing dried extract.

43. Mix thoroughly.

44. Transfer each sample to a column using glass pipettes.

45. Rinse sample vial twice, adding 300 µl of hexane each time, thus transferring a total volume of 900 µl to each column.

46. Wash each column twice with 900 µl of ethyl ether: hexane (30:70), allowing the flow-through to drip into the tray.

47. Wash each column twice with 750 µl of ethyl acetate: hexane (50:50), allowing the flow-through to drip into the tray.

48. Add 900 µl of ethyl ether: hexane (50:50) to each column and collect the flow-through in new 8-ml vials.

49. Repeat step 48 once, pooling both eluates in the same vial, for a total volume of 1.8 ml.

50. Discard the columns.

51. Concentrate the samples to dryness by vacuum centrifugation (~15 min) or under a nitrogen stream.

52. Add 300 µl of hexane to each sample and mix well to resuspend JH.

53. Evaporate the hexane under nitrogen gas (UHP grade) to ~25 µl of liquid.

54. Use a glass pipette to transfer the fluid into a tapered 250 µl glass vial insert (e.g. Agilent #5181-1270).

55. Repeat the steps 50-52 for obtaining a final concentrate volume of ~50 µl.

56. Using nitrogen, dry the liquid in the vial insert down to a final volume of 3 µl, measured with a graduated 10 µl syringe.

57. Using the syringe, repeatedly wash down the sides of the insert during the drying process to ensure all JH is within the remaining concentrate.

Repeated flushing through the syringe will also help evaporate down the last few microliters.

58. Syringes should be washed after each use by drawing up acetone then hexane (at least 5 times each).

59. Prepare the GC-MSD system for analysing samples:

   59.1. Install a Zebron ZB-Wax 30 m x 0.25 mm x 0.25 µm GC capillary column.
   59.2. Set initial temperature of GC to hold for 1 min at 60°C.
   59.3. Ramp the GC temperature up at 20°C / min to a final temperature of 240°C; hold for 20 min (i.e. a total run time of 30 min).
   59.4. Set the GC inlet protocol to pulsed splitless injection at 250°C.
   59.5. Set the pressure at 9.98 psi with a 23.8 ml/min flow rate for the carrier gas (UHP grade helium).
   59.6. Set the injection quantity to 1µl.
   59.7. Create a 5-min solvent delay.
   This prevents the recording of highly volatile solvents, which evaporate early on and can damage the detector.

60. Manually load the 1 µl of the concentrated sample using an injection syringe approved by the GC-MSD manufacturer.

61. To ensure accuracy, first draw up 1-2 µl of air, then the sample then another 1-2 µl of air.

62. To ensure specificity, monitor MSD results at m/x 76 and 225 for the JH-III derivative, and at 69, 84 and 136 for farnesol.

63. Measure the peak area for JH and adjust the value to account for any changes indicated by the results for farnesol, which was added in step 6 to serve as internal standard

64. Calculate the final titre based on the haemolymph volume and a standard curve.

The latter can be prepared with 5, 25, 125, 250 μg or more JH III, and starting the process at step 30.

 

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