*Note: An internal standard with known concentrations of deuterated forms of each target analyte in 40% MeOH will be required.
1. Label a centrifuge tube for each sample and at least three process blanks.
2. Add Lysing Matrix D beads (~300 mg) to each tube and then record weight of tube + beads.
3. Add powder sample (20-100 mg) to each tube and reweigh to calculate sample mass.
4. Add 1500 μl MeOH + 100 μl internal standard to each tube.
5. Vortex each tube for 60 seconds at 10 krpm and place in rotator.
6. Rotate at 30 rpm for 2 hours at 4°C.
7. Label an autosampler vial for each sample and process blank.
8. Centrifuge samples for 8 minutes at 14000 g Force.
9. Transfer 1350 μl of supernatant from each tube to the appropriately labeled vial.
*About 1 out of every 20 SLE columns we used had interference for testosterone and androstenedione in our analyses, so backup samples were helpful. If a backup sample/duplicate is desired the supernatant should be divided into 2 vials at this point, but remember that each sample (primary and backup) will contain only half the extracted analyte.
10. Evaporate samples to < 50 μl in vacuum concentrator (90-120 minutes, set to 5.1 torr, ambient temp).
11. Add 40 μl clean MeOH + 300 μl water to each vial.
12. Cap vials and vortex each for 20 seconds at 10 krpm to reconstitute.
Supported liquid extraction (SLE)
*Note: We pre-cooled the rotor from the vacuum concentrator for 3 hours at -20°C prior to use. A unit with refrigeration would make this step unneccessary.
13. Label an autosampler vial for each desired control sample, one SLE process blank, and one double blank.
14. For control samples, add 100 μl control sample + 100 μl internal standard + 200 μl water to each vial.
15. For SLE process blank, add 100 μl internal standard + 300 μl water.
16. For double blank, add 400 μl water.
17. Label an autosampler vial for each sample, process blank, and control sample.
18. Load labeled vials and SLE columns in positive pressure manifold.
19. Pipette each reconstituted sample, control sample, and blank into designated SLE column.
20. Apply 0.5-3.0 psi positive pressure (N2) to load sample into column (about 5 seconds).
21. Wait 5 minutes.
22. Add 1800 μl Methyl tert-Butyl Ether (MTBE) to each column.
23. Wait 10 minutes to allow passive gravity elution.
24. Apply 0.5-1.0 psi positive pressure (N2) for 5 seconds.
25. Wait 5 minutes.
26. Blow columns dry with applying pressure incrementally at 0.5psi, 3 psi, 6 psi, and 9 psi (10 seconds each).
27. Move vials with eluted MTBE to vacuum concentrator.
28. Evaporate samples to dryness in vacuum concentrator with precooled rotor (20-30 minutes, set to 5.1 torr, ambient temp).
29. Remove vials from concetrator and place on ice until after reconstituted.
30. Reconstitute samples by adding 200 μl 40% MeOH to each vial.
31. Cap vials and vortex each for 20 seconds at 10 krpm.
32. Store vials in freezer until LC-MS/MS analysis.