Sample preparation

After extracting the content out of your matrix, the sample preparation for LC-MS/GC-MS analysis can take place.

This protocol only briefly describes the sample preparation procedure. For detailed procedure see the Metamino^® user manual.

LC-MS analysis goes as followed:

1. Pipette 100 μL of your extracted content (serum, plasma, ...) and add 100 μL of precipitation medium. Centrifuge 30 to 60 sec at 1,500 ×g (6,000 rpm).
2. Pipette 25 μL of your precipitated sample and add 10 μL of solution with internal standard to each sample preparation vial.
3. Pipette 25 μL of catalytic solution into each sample preparation vial and vortex 5-10 sec.
4. Pipette 10 μL of reagent solution into each sample preparation vial and vortex 5-10 sec. Let the derivatization proceed for at least 2-3 minutes.
5. Activate and equilibrate the sorbent in microspin filter by adding:
   1. 200 μL of MSPE activation medium, then centrifuge 30 to 60 sec at 1,500 ×g (6,000 rpm).
   2. 200 μL of MSPE sorbent equilibration medium, then centrifuge 30 to 60 sec at 1,500 ×g (6,000 rpm).
6. Discard the flow through.
7. Dilute derivatization reaction mixture with 400 μL of diluting and washing medium and vortex 5-10 sec.
8. Load the diluted reaction mixture (typically 450-500 μL) to the wetted microspin filter sorbent and let it stand for 1-2 min. Centrifuge 30 to 60 sec at 1,500 ×g (6,000 rpm).
9. Discard flow through.
10. Wash the sorbent in the microspin filter with 200 μL of diluting and washing medium and centrifuge 30 to 60 sec at 1,500 ×g (6,000 rpm).
11. Place the microspin filter into a new centrifugal vial, add 200 μL of eluting medium and centrifuge 30 to 60 sec at 1,500 ×g (6,000 rpm).
12. Transfer the eluate into the autosampler vial. The sample is ready for LC-MS analysis.

GC-MS analysis goes as followed:

1. Pipette 25 μL of your target sample and 10 μL of solution with internal standards into each reaction glass tube.
2. Pipette 20 μL of reducing agent into each reaction glass tube and vortex briefly 5 to 10 sec. Let it stand for 1 to 2 minutes.
3. Pipette 25 μL of basic medium into each reaction glass tube and add 50 μL of reagent (derivatization) solution into each reaction glass tube and vortex 5 to 10 sec.
4. Pipette 50 μL of reagent (derivatization) solution medium into each reaction glass tube and vortex 5 to 10 sec.
5. Pipette 25 μL of catalytic solution into each sample preparation vial and vortex 5 to 10 sec.
6. Pipette 25 μL of catalytic solution into each sample preparation vial and vortex 5 to 10 sec. Let it stand for 1-2 minutes. The emulsion will gradually separate into two layers.
7. Pipette 50 μL of extraction medium into each sample preparation vial and vortex 5 to 10 sec.
8. Pipette 25 μL of acidic medium into each sample preparation vial and vortex 5 to 10 sec. Then centrifuge 30 to 60 sec 1,500 ×g (6,000 rpm).
9. Transfer the organic (upper) layer (50-100 µL) into the autosampler vial with insert. The sample is ready for GC-MS analysis.