Coenzyme Q10 in Serum/Plasma/Whole Blood - HPLC

Plasma/Serum

• Place 500 μl plasma/serum/whole blood and 250 μl Internal Standard into a labeled, light protected reaction vial and mix briefly.
• Add 500 μl Precipitation Reagent 1 and mix 30 s (vortex).
• Incubate 10 minutes at +2 to +8 °C.
• Centrifuge 5 minutes at 15 000 x g. It is not necessary to transfer the supernatant into a new reaction vial!
• Add 100 μl Precipitation Reagent 2 and mix 30 s (vortex).
• Centrifuge 10 minutes at 15 000 x g.
• Apply complete supernatant to Sample Clean Up Column and draw through completely by centrifugation (2 minutes at 700 x g) or suction, discard effluent.
• Draw 500 μl Wash Buffer 1 through the Sample Clean Up Column (1 minute at 700 x g), followed by 160 μl Wash Buffer 2 (2 minutes at 700 x g), discard effluents.
• Centrifuge 250 μl Elution Buffer through the Sample Clean up Column in a labeled, light protected reaction vial (2 min at 700 x g).
• Inject 50 μl of the eluate into the HPLC system.

Whole Blood

• Place 250 µl cold dist. water into a labelled, light protected reaction vial, add 250 µl whole blood and mix briefly. To make sure that the blood is transferred completely rinse the tip several times.
• Add 250 µl Internal Standard and mix briefly.
• Add 500 µl Precipitation Reagent 1 and mix for 30 sec (vortex).
• Incubate 10 min at +2 to +8 °C.
• Centrifuge 5 min at 15000 x g. It is not necessary to transfer the supernatant into a new reaction vial!
• Add 100 µl Precipitation Reagent 2 and mix for 30 sec (vortex).
• Centrifuge immediately for 10 min at 15000 x g.
• Apply complete supernatant from the precipitation step to a labelled sample clean up column and draw through quickly by centrifugation (2 min at 700 x g) or suction, discard effluent.
• Draw 500 µl Wash Buffer 1 through the column by centrifugation (1 min at 700 x g) or suction, discard effluent.
• Draw 160 µl Wash Buffer 2 through the column by centrifugation (2 min at 700 x g) or suction, discard effluent.
• Change the collection vial, apply 250 µl Elution Buffer to the column, and draw through completely by centrifugation (2 min at 700 x g) or suction.
• Inject 50 µl eluate into the HPLC system.