2.3. Preparation of solvents and HPLC standards
Our HPLC method is based on a previously published
method [26]. Mobile phase solvents were made by mixing
acetonitrile, tetrahydrofuran and methanol, then adding
1% ammonium sulfate in HPLC grade water and 0.0005%
-hydroxytoluene as an antioxidant. Solvent A contained
acetonitrile–tetrahydrofuran–methanol–1% aqueous ammonium
sulfate solution (85:5:5:5, v/v/v/v), and solvent B contained
acetonitrile–tetrahydrofuran–methanol–1% aqueous
ammonium sulfate solution (55:35:5:5, v/v/v/v). Each solvent
was prepared fresh before use. HPLC standards were
prepared by dissolving approximately 1mg standard into
10mL toluene–ethanol (1:99, v/v). Standards were prepared
by mass, and stored in the dark at 4 ◦C for up to two weeks
before use.