4.12. CoQ extraction for CoQ pool size determination

To extract CoQ from tissues, 5 mg frozen tissue was weighed into cooled lysis tubes (Precellys, CK-14) on dry ice. Then 150 μl KPi buffer (50 mM KPi, pH 7.8) was added and tissue was homogenised in the bullet blender (Bertin Instruments, Precellys 24) at setting 6500 during 15 s (CK14 (1.4 mm) beads). CoQ was extracted from 100 μl of the homogenate by adding a mixture of 250 μl ice-cold acidified methanol and 250 μl hexane and vortexing. The upper, CoQ-containing hexane layer was separated by centrifugation (5 min, 17,000×g, 4°C) and then dried down in 1 ml glass mass spectrometry vials (186005663CV, Waters) under a stream of N₂. Dried samples were then resuspended in methanol containing 2 mM ammonium formate. Heart tissue was diluted 1:10 (20 μl in 170 μl methanol containing 2 mM ammonium formate and 10 μl of 5 μM d₆-CoQ₁₀ internal standard), while liver homogenate was either not diluted (CoQ₁₀ analysis) or diluted 1:3 (CoQ₉ analysis). Samples were overlaid with argon and stored at −20°C until analysis.