It depends on what exactly you're analyzing for, but generally it is good to clean up the sample as much as possible to avoid the reagent reacting with hydroxyl groups on unrelated compounds in the sample, 100 uL/mg is about what I have used for cholesterol and oxysterols, gently heat such as on a steam bath for 15 minutes and quench with water, which also helps the partitioning to the sample solvent (hexane is what I use for sterols).

thank you Jonathan..=)