Measurement of CoQ10 in plasma and pancreatic tissue

The concentration of CoQ₁₀ in the plasma and pancreatic tissues was measured by LC-MS/MS analysis. CoQ₁₀ was extracted from the plasma and tissue and quantified using LC-MS/MS method reported previously [16], with some modifications. Given that CoQ₁₀ is an endogenous substance, all calibration and quality control samples were prepared in a physiological solution (4% bovine serum albumin in PBS). Briefly, 100 μL aliquots of rat plasma or tissue homogenates treated with 10 μL of CoQ₁₀ (800 ng/mL, as the internal standard) were extracted by liquid-liquid extraction using 1 mL isopropyl alcohol, followed by LC-MS/MS analysis. The samples were analyzed using a Shimadzu Nexera X2 UPLC (Shimadzu Corporation, Kyoto, Japan) coupled with an LCMS-8050 triple quadruple mass spectrometer (Shimadzu Corporation) with an electrospray ionization interface in the positive ion mode. Chromatographic separation was achieved using a Kinetex C18 column (2.1 × 100 mm, 2.6 μm; Phenomenex, Seoul, Korea) with a mobile phase consisting of 0.1% formic acid in isopropyl alcohol and methanol at a flow rate of 0.2 mL/min. The total run time was 4 minutes per sample. Quantitation was performed using selected reaction monitoring of the transitions at m/z 863.35 > 197.15 (for CoQ₁₀) and m/z 794.35 > 197.15 (for the internal standard). The calibration curves were linear (r ≥ 0.995) from 20 to 10,000 ng/mL. The within- and between-batch precision and accuracy were within the acceptable limits of ± 15%.