Figure 2. Variations of AcAc stability are matrix and temperature dependent, but AcAc is tolerant to freeze thaw cycles.

Stability of synthesized AcAc (n=3/group) in (A) water or (B) AcN/MeOH/water (2:2:1) extraction solution (n=3/group), stored at +4 C, 21 C (room temperature), +37 C, −20 C, −80 C, or LN₂, quantified by the AcAc/βOHB ratio (100 μM AcAc and 20 μM βOHB were added simultaneously during sample preparation; βOHB does not undergo decarboxylation and is stable). (C) Stability of endogenous AcAc in serum (n=3–6/group), stored at +4 C, room temperature, +37 C, −20 C, −80 C, or LN₂, quantified by AcAc/[3,4,4,4-D₄]βOHB (20 μM [3,4,4,4-D₄]βOHB was added to serum immediately after collection from mice fasted for 24h). (D) Stability of AcAc as calculated by the AcAc/βOHB (in water or AcN/MeOH/water) or AcAc/[3,4,4,4-D₄]βOHB (in serum) signal ratios in fresh samples and samples subjected to three freeze/thaw cycles, all relative to unthawed sample (n=6/group). Freeze/thaw cycles were performed within first nine hours of storage after adding 100 μM AcAc and 20 μM βOHB (in water or AcN/MeOH/water) or 20 μM [3,4,4,4-D₄]βOHB, serum from mice in the fasted state. Data expressed as the mean standard error of the mean (SEM).